Plant regulatory sequences for selective control of gene expression

ABSTRACT

Promoters from male reproductive tissues were isolated from corn ( Zea mays ). These promoters can be used in plants to regulate transcription of target genes including genes for control of fertility, insect or pathogen tolerance, herbicide tolerance or any gene of interest.

RELATED APPLICATION DATA

[0001] This application claims priority to U.S. Provisional Application60/201,255, filed on May 1, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to the isolation and use of nucleicacid molecules for control of gene expression in plants, specificallynovel plant promoters.

BACKGROUND OF THE INVENTION

[0003] One of the goals of plant genetic engineering is to produceplants with agronomically important characteristics or traits. Recentadvances in genetic engineering have provided the requisite tools totransform plants to contain and express foreign genes (Kahl et al. ,1995, World Journal of Microbiology and Biotechnology 11:449-460).Particularly desirable traits or qualities of interest for plant geneticengineering would include, but are not limited to, resistance toinsects, fungal diseases, and other pests and disease-causing agents,tolerances to herbicides, enhanced stability, yield, or shelf-life,environmental tolerances, and nutritional enhancements. Thetechnological advances in plant transformation and regeneration haveenabled researchers to take pieces of DNA, such as a gene or genes froma heterologous source, or a native source, but modified to havedifferent or improved qualities, and incorporate the exogenous DNA intothe plant's genome. The gene or gene(s) can then be expressed in theplant cell to exhibit the added characteristic(s) or trait(s). In oneapproach, expression of a novel gene that is not normally expressed in aparticular plant or plant tissue may confer a desired phenotypic effect.In another approach, transcription of a gene or part of a gene in anantisense orientation may produce a desirable effect by preventing orinhibiting expression of an endogenous gene.

[0004] Isolated plant promoters are useful for modifying plants throughgenetic engineering to have desired phenotypic characteristics. In orderto produce such a transgenic plant, a vector that includes aheterologous gene sequence that confers the desired phenotype whenexpressed in the plant is introduced into the plant cell. The vectoralso includes a plant promoter that is operably linked to theheterologous gene sequence, often a promoter not normally associatedwith the heterologous gene. The vector is then introduced into a plantcell to produce a transformed plant cell, and the transformed plant cellis regenerated into a transgenic plant. The promoter controls expressionof the introduced DNA sequence to which the promoter is operably linkedand thus affects the desired characteristic conferred by the DNAsequence.

[0005] Because the promoter is a regulatory element that plays anintegral part in the overall expression of a gene or gene(s), it wouldbe advantageous to have a variety of promoters to tailor gene expressionsuch that a gene or gene(s) is transcribed efficiently at the right timeduring plant growth and development, in the optimal location in theplant, and in the amount necessary to produce the desired effect. In onecase, for example, constitutive expression of a gene product may bebeneficial in one location of the plant, but less beneficial in anotherpart of the plant. In other cases, it may be beneficial to have a geneproduct produced at a certain developmental stage of the plant, or inresponse to certain environmental or chemical stimuli. The commercialdevelopment of genetically improved germplasm has also advanced to thestage of introducing multiple traits into crop plants, often referred toas a gene stacking approach. In this approach, multiple genes conferringdifferent characteristics of interest can be introduced into a plant. Itis important when introducing multiple genes into a plant, that eachgene is modulated or controlled for optimal expression and that theregulatory elements are diverse, to reduce the potential of genesilencing that can be caused by recombination of homologous sequences.In light of these and other considerations, it is apparent that optimalcontrol of gene expression and regulatory element diversity areimportant in plant biotechnology.

[0006] The proper regulatory sequences must be present and in the properlocation with respect to the DNA sequence of interest for the newlyinserted DNA to be transcribed and thereby, if desired, translated intoa protein in the plant cell. These regulatory sequences include, but arenot limited to, a promoter, a 5′ untranslated leader, and a 3′polyadenylation sequence. The ability to select the tissues in which totranscribe such foreign DNA and the time during plant growth in which toobtain transcription of such foreign DNA is also possible through thechoice of appropriate promoter sequences that control transcription ofthese genes.

[0007] A variety of different types or classes of promoters can be usedfor plant genetic engineering. Promoters can be classified on the basisof range or tissue specificity. For example, promoters referred to asconstitutive promoters are capable of transcribing operatively linkedDNA sequences efficiently and expressing said DNA sequences in multipletissues. Tissue-enhanced or tissue-specific promoters can be foundupstream and operatively linked to DNA sequences normally transcribed inhigher levels in certain plant tissues or specifically in certain planttissues. Other classes of promoters would include, but are not limitedto, inducible promoters that can be triggered by external stimuli suchas chemical agents, developmental stimuli, or environmental stimuli.Thus, the different types of promoters desired can be obtained byisolating the regulatory regions of DNA sequences that are transcribedand expressed in a constitutive, tissue-enhanced, or inducible manner.

[0008] The technological advances of high-throughput sequencing andbioinformatics has provided additional molecular tools for promoterdiscovery. Particular target plant cells, tissues, or organs at aspecific stage of development, or under particular chemical,environmental, or physiological conditions can be used as sourcematerial to isolate the mRNA and construct cDNA libraries. The cDNAlibraries are quickly sequenced, and the expressed sequences can becatalogued electronically. Using sequence analysis software, thousandsof sequences can be analyzed in a short period, and sequences fromselected cDNA libraries can be compared. The combination of laboratoryand computer-based subtraction methods allows researchers to scan andcompare cDNA libraries and identify sequences with a desired expressionprofile. For example, sequences expressed preferentially in one tissuecan be identified by comparing a cDNA library from one tissue to cDNAlibraries of other tissues and electronically “subtracting” commonsequences to find sequences only expressed in the target tissue ofinterest. The tissue enhanced sequence can then be used as a probe orprimer to clone the corresponding full-length cDNA. A genomic library ofthe target plant can then be used to isolate the corresponding gene andthe associated regulatory elements, including but not limited topromoter sequences.

[0009] Multiple genes that have a desired expression profile such as inmale reproductive tissues can be isolated by selectively comparing cDNAlibraries of target tissues of interest with non-target or backgroundcDNA libraries to find the 5′ regulatory regions associated with theexpressed sequences in those target libraries. The promoter sequencescan be isolated from the genomic DNA flanking the desired genes. Theisolated promoter sequences can be used for selectively modulatingexpression of any operatively linked gene and provide additionalregulatory element diversity in a plant expression vector in genestacking approaches.

SUMMARY OF THE INVENTION

[0010] The present invention provides isolated plant promoter sequencesthat comprise nucleic acid regions located upstream of the 5′ end ofplant DNA structural coding sequences that are transcribed in malereproductive tissues. The plant promoter sequences are capable ofmodulating or initiating transcription of DNA sequences to which theyare operably linked.

[0011] The present invention provides nucleic acid sequences comprisingregulatory sequences as shown in SEQ ID NOS: 79-98 that are locatedupstream of the 5′ end of plant DNA structural coding sequences andtranscribed in male reproductive tissues.

[0012] In one aspect, the present invention provides nucleic acidsequences comprising a sequence selected from the group consisting ofSEQ ID NOS: 79-98 or any fragments or regions of the sequence or ciselements of the sequence that are capable of regulating transcription ofoperably linked DNA sequences.

[0013] The present invention also provides nucleic acid sequencescomprising a sequence selected from the group consisting of SEQ ID NOS:79-98 that are promoters.

[0014] Another aspect of the present invention relates to the use of oneor more cis elements, or fragments thereof of the disclosed 5′ promotersequences that can be combined to create novel promoters or used in anovel combination with another heterologous regulatory sequence tocreate a chimeric promoter capable of modulating transcription of anoperably linked DNA sequence.

[0015] Hence, the present invention relates to the use of nucleic acidsequences disclosed in SEQ ID NOS: 79-98 or any fragment, region, or ciselement of the disclosed sequences that are capable of regulatingtranscription of a DNA sequence when operably linked to the DNAsequence. Therefore, the invention not only encompasses the sequences asdisclosed in SEQ ID NOS: 79-98, but also includes any truncated ordeletion derivatives, or fragments or regions thereof that are capableof functioning independently as a promoter including cis elements thatare capable of functioning as regulatory sequences in conjuction withone or more regulatory sequences when operably linked to a transcribablesequence.

[0016] The present invention thus encompasses a novel promoter orchimeric or hybrid promoter comprising a nucleic acid of SEQ ID NOS:79-98. The chimeric or hybrid promoters can consist of any lengthfragments, regions, or cis elements of the disclosed sequences of SEQ IDNOS: 79-98 combined with any other transcriptionally active minimal orfull-length promoter. For example, a promoter sequence selected from SEQID NOS: 79-98 may be combined with a CaMV 35S or other promoter toconstruct a novel chimeric promoter. A minimal promoter can also be usedin combination with the nucleic acid sequences of the present invention.A novel promoter also comprises any promoter constructed by engineeringthe nucleic acid sequences disclosed in SEQ ID NOS: 79-98 or anyfragment, region, or cis element of the disclosed sequences in anymanner sufficient to transcribe an operably linked DNA sequence.

[0017] Another aspect of the present invention relates to the ability ofthe promoter sequences of SEQ ID NOS: 79-98, or fragments, regions, orcis elements thereof to regulate transcription of operably linkedtranscribable sequences in male reproductive tissues. Fragments,regions, or cis elements of SEQ ID NOS: 79-98 that are capable ofregulating transcription of operably linked DNA sequences in certaintissues may be isolated from the disclosed nucleic acid sequences of SEQID NOS: 79-98 and used to engineer novel promoters.

[0018] The present invention also encompasses DNA constructs comprisingthe disclosed sequences as shown in SEQ ID NOS: 79-98 or any fragments,regions, or cis elements thereof, including novel promoters generatedusing the disclosed sequences or any fragment, region, or cis element ofthe disclosed sequences.

[0019] The present invention also includes any transgenic cells andplants containing the DNA disclosed in the sequences as shown in SEQ IDNOS: 79-98, or any fragments, regions, or cis elements thereof.

[0020] The present invention also provides a method of regulatingtranscription of a DNA sequence comprising operably linking the DNAsequence to any promoter comprising a nucleic acid comprising all or anyfragment, region or cis element of a sequence selected from the groupconsisting of SEQ ID NOS: 79-98.

[0021] In another embodiment the present invention provides a method ofregulating expression of DNA sequences in male reproductive tissues byoperably linking a sequence selected from the group consisting of SEQ IDNOS: 79-98, or any fragment, region, or cis element of the disclosedsequences to any transcribable DNA sequence. The fragments, regions, orcis elements of the disclosed promoters as shown in SEQ ID NOS: 79-98can be engineered and used independently in novel combinations includingmultimers, or truncated derivatives and the novel promoters can beoperably linked with a transcribable DNA sequence. Alternatively thedisclosed fragments, regions, or cis elements of the disclosed sequencescan be used in combination with a heterologous promoter including aminimal promoter to create a novel chimeric or hybrid promoter and thenovel chimeric promoter can be operably linked to a transcribable DNAsequence.

[0022] The present invention also provides a method of making atransgenic plant by introducing into a cell of a plant a DNA constructcomprising: (i) a promoter comprising a nucleic acid comprising asequence selected from the group consisting of SEQ ID NOS: 79-98, orfragment, region, or cis element thereof, and operably linked to thepromoter, (ii) a transcribable DNA sequence and (iii) a 3′ untranslatedregion.

[0023] The present invention also provides a method of isolating atleast one 5′ regulatory sequence of a desired expression profile from atarget plant of interest by evaluating a collection of nucleic acidsequences of ESTs derived from one or more cDNA libraries prepared froma plant cell type of interest, comparing EST sequences from at least onetarget plant cDNA library and one or more non-target cDNA libraries ofESTs from a different plant cell type, subtracting common EST sequencesfound in both target and non-target libraries, designing gene-specificprimers from the remaining ESTs after the subtraction that arerepresentative of the targeted expressed sequences, and isolating thecorresponding 5′ flanking and regulatory sequences, that includespromoter sequences from a genomic library prepared from the target plantusing the gene specific primers.

[0024] The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a plasmid map of pMON19469.

[0026]FIG. 2 is a plasmid map of pMON51850.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0027] Seq ID NOs: 1-3 are adaptor sequences.

[0028] Seq ID NOs: 4-78 are fully synthesized primers derived from knownZea mays sequences.

[0029] Seq ID NOs: 79-98 are promoter sequences isolated from Zea mays.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Definitions and Methods

[0031] The following definitions and methods are provided to betterdefine the present invention and to guide those of ordinary skill in theart in the practice of the present invention. Unless otherwise noted,terms are to be understood according to conventional usage by those ofordinary skill in the relevant art. The nomenclature for DNA bases asset forth at 37 CFR §1.822 is used. The standard one- and three-letternomenclature for amino acid residues is used.

[0032] “Nucleic acid (sequence)” or “polynucleotide (sequence)” refersto single- or double-stranded DNA or RNA of genomic or synthetic origin,i.e., a polymer of deoxyribonucleotide or ribonucleotide bases,respectively, read from the 5′ (upstream) end to the 3′ (downstream)end. The nucleic acid can represent the sense or complementary(antisense) strand.

[0033] “Native” refers to a naturally occurring (“wild-type”) nucleicacid sequence.

[0034] “Heterologous” sequence refers to a sequence that originates froma foreign source or species or, if from the same source, is modifiedfrom its original form.

[0035] An “isolated” nucleic acid sequence is substantially separated orpurified away from other nucleic acid sequences that the nucleic acid isnormally associated with in the cell of the organism in which thenucleic acid naturally occurs, i.e., other chromosomal orextrachromosomal DNA. The term embraces nucleic acids that arebiochemically purified so as to substantially remove contaminatingnucleic acids and other cellular components. The term also embracesrecombinant nucleic acids and chemically synthesized nucleic acids.

[0036] The term “substantially purified”, as used herein, refers to amolecule separated from substantially all other molecules normallyassociated with it in its native state. More preferably, a substantiallypurified molecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, preferably75% free, more preferably 90% free from the other molecules (exclusiveof solvent) present in the natural mixture. The term “substantiallypurified” is not intended to encompass molecules present in their nativestate.

[0037] A first nucleic acid sequence displays “substantial identity” toa reference nucleic acid sequence if, when optimally aligned (withappropriate nucleotide insertions or deletions totaling less than 20percent of the reference sequence over the window of comparison) withthe other nucleic acid (or its complementary strand), there is at leastabout 75% nucleotide sequence identity, preferably at least about 80%identity, more preferably at least about 85% identity, and mostpreferably at least about 90% identity over a comparison window of atleast 20 nucleotide positions, preferably at least 50 nucleotidepositions, more preferably at least 100 nucleotide positions, and mostpreferably over the entire length of the first nucleic acid. Optimalalignment of sequences for aligning a comparison window may be conductedby the local homology algorithm of Smith and Waterman (Adv. Appl. Math.2: 482, 1981); by the homology alignment algorithm of Needleman andWunsch (J. Mol. Biol. 48:443, 1970); by the search for similarity methodof Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988);preferably by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software PackageRelease 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.).The reference nucleic acid may be a full-length molecule or a portion ofa longer molecule. Alternatively, two nucleic acids have substantialidentity if one hybridizes to the other under stringent conditions, asdefined below.

[0038] A first nucleic acid sequence is “operably linked” with a secondnucleic acid sequence when the sequences are so arranged that the firstnucleic acid sequence affects the function of the second nucleic acidsequence. Preferably, the two sequences are part of a single contiguousnucleic acid molecule and more preferably are adjacent. For example, apromoter is operably linked to a gene if the promoter regulates ormediates transcription of the gene in a cell.

[0039] A “recombinant” nucleic acid is made by an artificial combinationof two otherwise separated segments of sequence, e.g., by chemicalsynthesis or by the manipulation of isolated segments of nucleic acidsby genetic engineering techniques. Techniques for nucleic-acidmanipulation are well-known (see, e.g., Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, Sambrook et al., 1989;Current Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing and Wiley-Interscience, New York, 1992, with periodicupdates, Ausubel et al., 1992; and PCR Protocols: A Guide to Methods andApplications, Academic Press: San Diego, Innis et al., 1990). Methodsfor chemical synthesis of nucleic acids are discussed, for example, inBeaucage and Carruthers (Tetra. Letts. 22:1859-1862, 1981), andMatteucci et al. (J. Am. Chem. Soc. 103:3185, 1981). Chemical synthesisof nucleic acids can be performed, for example, on commercial automatedoligonucleotide synthesizers.

[0040] A “synthetic nucleic acid sequence” can be designed andchemically synthesized for enhanced expression in particular host cellsand for the purposes of cloning into appropriate vectors. Host cellsoften display a preferred pattern of codon usage (Murray et al., 1989).Synthetic DNAs designed to enhance expression in a particular hostshould therefore reflect the pattern of codon usage in the host cell.Computer programs are available for these purposes including but notlimited to the “BestFit” or “Gap” programs of the Sequence AnalysisSoftware Package, Genetics Computer Group, Inc. (University of WisconsinBiotechnology Center, Madison, Wis. 53711).

[0041] “Amplification” of nucleic acids or “nucleic acid reproduction”refers to the production of additional copies of a nucleic acid sequenceand is carried out using polymerase chain reaction (PCR) technologies. Avariety of amplification methods are known in the art and are described,inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and by Innis etal. (PCR Protocols: A Guide to Methods and Applications, Academic Press,San Diego, 1990). In PCR, a primer refers to a short oligonucleotide ofdefined sequence that is annealed to a DNA template to initiate thepolymerase chain reaction.

[0042] “Transformed”, “transfected”, or “transgenic” refers to a cell,tissue, organ, or organism into which has been introduced a foreignnucleic acid, such as a recombinant vector. Preferably, the introducednucleic acid is integrated into the genomic DNA of the recipient cell,tissue, organ or organism such that the introduced nucleic acid isinherited by subsequent progeny. A “transgenic” or “transformed” cell ororganism also includes progeny of the cell or organism and progenyproduced from a breeding program employing such a “transgenic” plant asa parent in a cross and exhibiting an altered phenotype resulting fromthe presence of a recombinant construct or vector.

[0043] The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA,synthetic DNA, or other DNA that encodes a peptide, polypeptide,protein, or RNA molecule, and regions flanking the coding sequenceinvolved in the regulation of expression. Some genes can be transcribedinto mRNA and translated into polypeptides (structural genes); othergenes can be transcribed into RNA (e.g., rRNA, tRNA); and other types ofgenes function as regulators of expression (regulator genes).

[0044] “Expression” of a gene refers to the transcription of a gene toproduce the corresponding mRNA and translation of this mRNA to producethe corresponding gene product, i.e., a peptide, polypeptide, orprotein. Gene expression is controlled or modulated by regulatoryelements including 5′ regulatory elements such as promoters.

[0045] “Genetic component” refers to any nucleic acid sequence orgenetic element that may also be a component or part of an expressionvector. Examples of genetic components include, but are not limited to,promoter regions, 5′ untranslated leaders, introns, genes, 3′untranslated regions, and other regulatory sequences or sequences thataffect transcription or translation of one or more nucleic acidsequences.

[0046] The terms “recombinant DNA construct”, “recombinant vector”,“expression vector” or “expression cassette” refer to any agent such asa plasmid, cosmid, virus, BAC (bacterial artificial chromosome),autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence,derived from any source, capable of genomic integration or autonomousreplication, comprising a DNA molecule in which one or more DNAsequences have been linked in a functionally operative manner.

[0047] “Complementary” refers to the natural association of nucleic acidsequences by base-pairing (A-G-T pairs with the complementary sequenceA-C-T). Complementarity between two single-stranded molecules may bepartial, if only some of the nucleic acids pair are complementary, orcomplete, if all bases pair are complementary. The degree ofcomplementarity affects the efficiency and strength of hybridization andamplification reactions.

[0048] “Homology” refers to the level of similarity between nucleic acidor amino acid sequences in terms of percent nucleotide or amino acidpositional identity, respectively, i.e., sequence similarity oridentity. Homology also refers to the concept of similar functionalproperties among different nucleic acids or proteins.

[0049] “ESTs” or Expressed Sequence Tags are short sequences of randomlyselected clones from a cDNA (or complementary DNA) library that arerepresentative of the cDNA inserts of these randomly selected clones(McCombie et al., Nature Genetics, 1:124, 1992; Kurata et al., NatureGenetics, 8: 365,1994; Okubo et al., Nature Genetics, 2: 173, 1992).

[0050] The term “electronic Northern” refers to a computer-basedsequence analysis that allows sequences from multiple cDNA libraries tobe compared electronically based on parameters the researcher identifiesincluding abundance in EST populations in multiple cDNA libraries, orexclusively to EST sets from one or combinations of libraries.

[0051] “Subsetting” refers to a method of comparing nucleic acidsequences from different or multiple sources that can be used to assessthe expression profile of the nucleic acid sequences that reflects genetranscription activity and message stability in a particular tissue, ata particular time, or under particular conditions.

[0052] “Promoter” refers to a nucleic acid sequence located upstream or5′ to a translational start codon of an open reading frame (orprotein-coding region) of a gene and that is involved in recognition andbinding of RNA polymerase II and other proteins (trans-actingtranscription factors) to initiate transcription. A “plant promoter” isa native or non-native promoter that is functional in plant cells.Constitutive promoters are functional in most or all tissues of a plantthroughout plant development. Tissue-, organ- or cell-specific promotersare expressed only or predominantly in a particular tissue, organ, orcell type, respectively. Rather than being expressed “specifically” in agiven tissue, organ, or cell type, a promoter may display “enhanced”expression, i.e., a higher level of expression, in one part (e.g., celltype, tissue, or organ) of the plant compared to other parts of theplant. Temporally regulated promoters are functional only orpredominantly during certain periods of plant development or at certaintimes of day, as in the case of genes associated with circadian rhythm,for example. Inducible promoters selectively express an operably linkedDNA sequence in response to the presence of an endogenous or exogenousstimulus, for example, by chemical compounds (chemical inducers) or inresponse to environmental, hormonal, chemical, or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding, or chemicals such as ethanol, jasmonate, salicylic acid, orsafeners.

[0053] Any plant promoter can be used as a 5′ regulatory sequence formodulating expression of a particular gene or genes. One preferredpromoter would be a plant RNA polymerase II promoter. Plant RNApolymerase II promoters, like those of other higher eukaryotes, havecomplex structures and are comprised of several distinct elements. Onesuch element is the TATA box or Goldberg-Hogness box, which is requiredfor correct expression of eukaryotic genes in vitro and accurate,efficient initiation of transcription in vivo. The TATA box is typicallypositioned at approximately −25 to −35, that is, at 25 to 35 basepairs(bp) upstream (5′) of the transcription initiation site, or cap site,which is defined as position +1 (Breathnach and Chambon, Ann. Rev.Biochem. 50:349-383, 1981; Messing et al., In: Genetic Engineering ofPlants, Kosuge et al., eds., pp. 211-227, 1983). Another common element,the CCAAT box, is located between −70 and −100 bp. In plants, the CCAATbox may have a different consensus sequence than the functionallyanalogous sequence of mammalian promoters (the plant analogue has beentermed the “AGGA box” to differentiate it from its animal counterpart;Messing et al., In: Genetic Engineering of Plants, Kosuge et al., eds.,pp. 211-227, 1983). In addition, virtually all promoters includeadditional upstream activating sequences or enhancers (Benoist andChambon, Nature 290: 304-310, 1981; Gruss et al., Proc. Natl. Acad. Sci.USA 78:943-947, 1981; and Khoury and Gruss, Cell 27:313-314, 1983)extending from around −100 bp to −1,000 bp or more upstream of thetranscription initiation site. Enhancers have also been found 3′ to thetranscriptional start site.

[0054] When fused to heterologous DNA sequences, such promoterstypically cause the fused sequence to be transcribed in a manner that issimilar to that of the gene sequence that the promoter is normallyassociated with. Promoter fragments that include regulatory sequencescan be added (for example, fused to the 5′ end of, or inserted within,an active promoter having its own partial or complete regulatorysequences (Fluhr et al., Science 232:1106-1112, 1986; Ellis et al., EMBOJ. 6:11-16, 1987; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988;Comai et al., Plant Mol. Biol. 15:373-381, 1991). Alternatively,heterologous regulatory sequences can be added to the 5′ upstream regionof an inactive, truncated promoter, e.g., a promoter including only thecore TATA and, sometimes, the CCAAT elements (Fluhr et al., Science232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).

[0055] Promoters are typically comprised of multiple distinct“cis-acting transcriptional regulatory elements,” or simply“cis-elements,” each of which appears to confer a different aspect ofthe overall control of gene expression (Strittmatter and Chua, Proc.Nat. Acad. Sci. USA 84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16,1987; Benfey et al., EMBO J. 9:1677-1684, 1990). Cis elements bindtrans-acting protein factors that regulate transcription. Some ciselements bind more than one factor, and trans-acting transcriptionfactors may interact with different affinities with more than one ciselement (Johnson and McKnight, Ann. Rev. Biochem. 58:799-839, 1989).Plant transcription factors, corresponding cis elements, and analysis oftheir interaction are discussed, for example, in Martin (Curr. OpinionsBiotech. 7:130-138, 1996), Murai (Methods in Plant Biochemistry andMolecular Biology, Dashek, ed., CRC Press, 1997, pp. 397-422), andMaliga et al. (Methods in Plant Molecular Biology, Cold Spring HarborPress, 1995, pp. 233-300). The promoter sequences of the presentinvention can contain “cis elements” that can confer or modulate geneexpression.

[0056] Cis elements can be identified by a number of techniques,including deletion analysis, i.e., deleting one or more nucleotides fromthe 5′ end or internal to a promoter; DNA binding protein analysis usingDnase I footprinting; methylation interference; electrophoresismobility-shift assays, in vivo genomic footprinting by ligation-mediatedPCR; and other conventional assays; or by sequence similarity with knowncis element motifs by conventional sequence comparison methods. The finestructure of a cis element can be further studied by mutagenesis (orsubstitution) of one or more nucleotides or by other conventionalmethods (see for example, Methods in Plant Biochemistry and MolecularBiology, Dashek, ed., CRC Press, 1997, pp. 397-422; and Methods in PlantMolecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995,pp. 233-300).

[0057] Cis elements can be obtained by chemical synthesis or by cloningfrom promoters that include such elements, and they can be synthesizedwith additional flanking sequences that contain useful restrictionenzyme sites to facilitate subsequent manipulation. In one embodiment,the promoters are comprised of multiple distinct “cis-actingtranscriptional regulatory elements,” or simply “cis-elements,” each ofwhich appears to confer a different aspect of the overall control ofgene expression (Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987; Benfey et al.,EMBO J. 9:1677-1684, 1990). In a preferred embodiment, sequence regionscomprising “cis elements” of the nucleic acid sequences of SEQ ID NOS:79-98 are identified using computer programs designed specifically toidentify cis elements, or domains or motifs within sequences.

[0058] The present invention includes cis elements of SEQ ID NOS: 79-98,or homologues of cis elements known to affect gene regulation that showhomology with the nucleic acid sequences of the present invention. Anumber of such elements are known in the literature, such as elementsthat are regulated by numerous factors such as light, heat, or stress;elements that are regulated or induced by pathogens or chemicals, andthe like. Such elements may either positively or negatively regulatedgene expression, depending on the conditions. Examples of cis elementswould include, but are not limited to, oxygen responsive elements (Cowenet al., J. Biol. Chem. 268(36):26904, 1993), light regulatory elements(see for example, Bruce and Quaill, Plant Cell 2(11): 1081, 1990; andBruce et al., EMBO J. 10:3015, 1991), a cis element reponsive to methyljasmonate treatment (Beaudoin and Rothstein, Plant Mol. Biol. 33:835,1997), salicylic acid responsive elements (Strange et al., Plant J.11:1315, 1997), heat shock response elements (Pelham et al., TrendsGenet. 1:31, 1985), elements responsive to wounding and abiotic stress(Loace et al., Proc. Natl. Acad. Sci. U.S.A. 89:9230, 1992; Mhiri etal., Plant Mol. Biol. 33:257, 1997), low temperature elements (Baker etal., Plant Mol. Biol. 24:701, 1994; Jiang et al., Plant Mol. Biol.30:679, 1996; Nordin et al., Plant Mol. Biol. 21:641, 1993; Zhou et al.,J. Biol. Chem. 267:23515, 1992), and drought responsive elements,(Yamaguchi et al., Plant Cell 6:251-264, 1994; Wang et al., Plant Mol.Biol. 28:605, 1995; Bray, Trends in Plant Science 2:48, 1997).

[0059] The present invention therefore encompasses fragments or ciselements of the disclosed nucleic acid molecules, and such nucleic acidfragments can include any region of the disclosed sequences. Thepromoter regions or partial promoter regions of the present invention asshown in SEQ ID NOS: 79-98 can contain one or more regulatory elementsincluding but not limited to cis elements or domains that are capable ofregulating expression of operably linked DNA sequences, preferably inmale reproductive tissues.

[0060] Plant promoters can include promoters produced through themanipulation of known promoters to produce synthetic, chimeric, orhybrid promoters. Such promoters can also combine cis elements from oneor more promoters, for example, by adding a heterologous regulatorysequence to an active promoter with its own partial or completeregulatory sequences (Ellis et al., EMBO J. 6:11-16, 1987; Strittmatterand Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Poulsen andChua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., Plant. Mol. Biol.15:373-381, 1991). Chimeric promoters have also been developed by addinga heterologous regulatory sequence to the 5′ upstream region of aninactive, truncated promoter, i.e., a promoter that includes only thecore TATA and, optionally, the CCAAT elements (Fluhr et al., Science232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).

[0061] The design, construction, and use of chimeric or hybrid promoterscomprising one or more of cis elements of SEQ ID NOS: 79-98 formodulating or regulating the expression of operably linked nucleic acidsequences is also encompassed by the present invention.

[0062] The promoter sequences, fragments, regions or cis elementsthereof of SEQ ID NOS: 79-98 are capable of transcribing operably linkedDNA sequences in male reproductive tissues and therefore can selectivelyregulate expression of genes in these tissues.

[0063] The promoter sequences of the present invention are useful forregulating gene expression in male reproductive tissues such as tassels,anthers, and pollen. For a number of agronomic traits, transcription ofa gene or genes of interest is desirable in multiple tissues in order toconfer the desired characteristic(s). The availability of suitablepromoters that regulate transcription of operably linked genes inselected target tissues of interest is important because it may not bedesirable to have expression of a gene in every tissue, but only incertain tissues. For example, if one desires to selectively express atarget gene for controlling fertility in corn, it would be advantageousto have a promoter that confers enhanced expression in reproductivetissues. The promoter sequences of the present invention are capable ofregulating operably linked DNA sequence particularly in malereproductive tissues and have utility for regulating transcription ofany target gene including, but not limited to, genes for control offertility, insect or pathogen tolerance, herbicide tolerance or any geneof interest. Consequently, it is important to have a wide variety ofchoices of 5′ regulatory elements for any plant biotechnology strategy.

[0064] The advent of genomics, which comprises molecular andbioinformatics techniques, has resulted in rapid sequencing and analysesof a large number of DNA samples from a vast number of targets,including but not limited to plant species of agronomic importance. Toidentify the nucleic acid sequences of the present invention from adatabase or collection of cDNA sequences, the first step involvesconstructing cDNA libraries from specific plant tissue targets ofinterest. Briefly, the cDNA libraries are first constructed from thesetissues that are harvested at a particular developmental stage or underparticular environmental conditions. By identifying differentiallyexpressed genes in plant tissues at different developmental stages orunder different conditions, the corresponding regulatory sequences ofthose genes can be identified and isolated. Transcript imaging enablesthe identification of tissue-preferred sequences based on specificimaging of nucleic acid sequences from a cDNA library. By transcriptimaging as used herein is meant an analysis that compares the abundanceof expressed genes in one or more libraries. The clones contained withina cDNA library are sequenced and the sequences compared with sequencesfrom publicly available databases. Computer-based methods allow theresearcher to provide queries that compare sequences from multiplelibraries. The process enables quick identification of clones ofinterest compared with conventional hybridization subtraction methodsknown to those of skill in the art.

[0065] Using conventional methodologies, cDNA libraries can beconstructed from the mRNA (messenger RNA) of a given tissue or organismusing poly dT primers and reverse transcriptase (Efstratiadis et al.,Cell 7:279, 1976; Higuchi et al., Proc. Natl. Acad. Sci. U.S.A. 73:3146,1976; Maniatis et al., Cell 8:163, 1976; Land et al., Nucleic Acids Res.9:2251, 1981; Okayama et al., Mol. Cell. Biol. 2:161, 1982; Gubler etal., Gene 25:263, 1983).

[0066] Several methods can be employed to obtain full-length cDNAconstructs. For example, terminal transferase can be used to addhomopolymeric tails of dC residues to the free 3′ hydroxyl groups (Landet al., Nucleic Acids Res. 9:2251, 1981). This tail can then behybridized by a poly dG oligo that can act as a primer for the synthesisof full length second strand cDNA. Okayama and Berg, reported a methodfor obtaining full-length cDNA constructs (Mol. Cell Biol. 2:161, 1982).This method has been simplified by using synthetic primer adapters thathave both homopolymeric tails for priming the synthesis of the first andsecond strands and restriction sites for cloning into plasmids(Coleclough et al., Gene 34:305, 1985) and bacteriophage vectors(Krawinkel et al., Nucleic Acids Res. 14:1913, 1986; Han et al., NucleicAcids Res. 15:6304, 1987).

[0067] These strategies can be coupled with additional strategies forisolating rare mRNA populations. For example, a typical mammalian cellcontains between 10,000 and 30,000 different mRNA sequences (Davidson,Gene Activity in Early Development, 2nd ed., Academic Press, New York,1976). The number of clones required to achieve a given probability thata low-abundance mRNA will be present in a cDNA library isN=(1n(1−P))/(1n(1−1/n)) where N is the number of clones required, P isthe probability desired, and 1/n is the fractional proportion of thetotal mRNA that is represented by a single rare mRNA (Sambrook et al.,1989).

[0068] One method to enrich preparations of mRNA for sequences ofinterest is to fractionate by size. One such method is to fractionate byelectrophoresis through an agarose gel (Pennica et al., Nature 301:214,1983). Another method employs sucrose gradient centrifugation in thepresence of an agent, such as methylmercuric hydroxide, that denaturessecondary structure in RNA (Schweinfest et al., Proc. Natl. Acad. Sci.U.S.A. 79:4997-5000, 1982).

[0069] A frequently adopted method is to construct equalized ornormalized cDNA libraries (Ko, Nucleic Acids Res. 18:5705, 1990;Patanjali et al., Proc. Natl. Acad. Sci. U.S.A. 88:1943, 1991).Typically, the cDNA population is normalized by subtractivehybridization (Schmid et al., J. Neurochem. 48:307, 1987; Fargnoli etal., Anal. Biochem. 187:364, 1990; Travis et al., Proc. Natl. Acad. Sci.U.S.A. 85:1696, 1988; Kato, Eur. J. Neurosci. 2:704, 1990; Schweinfestet al., Genet. Anal. Tech. Appl. 7:64, 1990). Subtraction representsanother method for reducing the population of certain sequences in thecDNA library (Swaroop et al., Nucleic Acids Res. 19:1954, 1991).Normalized libraries can be constructed using the Soares procedure(Soares et al., Proc. Natl. Acad. Sci. U.S.A. 91:9228, 1994). Thisapproach is designed to reduce the initial 10,000-fold variation inindividual cDNA frequencies to achieve abundance within one order ofmagnitude while maintaining the overall sequence complexity of thelibrary. In the normalization process, the prevalence of high-abundancecDNA clones decreases dramatically, clones with mid-level abundance arerelatively unaffected, and clones for rare transcripts are effectivelyincreased in abundance.

[0070] ESTs can be sequenced by a number of methods. Two basic methodscan be used for DNA sequencing, the chain termination method (Sanger etal., Proc. Natl. Acad. Sci. U.S.A. 74: 5463, 1977) and the chemicaldegradation method (Maxam and Gilbert, Proc. Nat. Acad. Sci. U.S.A. 74:560, 1977). Automation and advances in technology, such as thereplacement of radioisotopes with fluorescence-based sequencing, havereduced the effort required to sequence DNA (Craxton, Methods, 2: 20,1991; Ju et al., Proc. Natl. Acad. Sci. U.S.A. 92: 4347, 1995; Tabor andRichardson, Proc. Natl. Acad. Sci. U.S.A. 92: 6339, 1995). Automatedsequencers are available from a number of manufacturers includingPharmacia Biotech, Inc., Piscataway, N.J. (Pharmacia ALF); LI-COR, Inc.,Lincoln, Nebr. (LI-COR 4,000); and Millipore, Bedford, Mass. (MilliporeBaseStation).

[0071] ESTs longer than 150 bp have been found to be useful forsimilarity searches and mapping (Adams et al., Science 252:1651, 1991).EST sequences normally range from 150-450 bases. This is the length ofsequence information that is routinely and reliably generated usingsingle run sequence data. Typically, only single run sequence data isobtained from the cDNA library (Adams et al., Science 252:1651, 1991).Automated single run sequencing typically results in an approximately2-3% error or base ambiguity rate (Boguski et al., Nature Genetics,4:332, 1993).

[0072] EST databases have been constructed or partially constructedfrom, for example, C. elegans (McCombrie et al., Nature Genetics 1:124,1992); human liver cell line HepG2 (Okubo et al., Nature Genetics 2:173,1992); human brain RNA (Adams et al., Science 252:1651, 1991; Adams etal., Nature 355:632, 1992); Arabidopsis, (Newman et al., Plant Physiol.106:1241, 1994); and rice (Kurata et al., Nature Genetics 8:365, 1994).The present invention uses ESTs from a number of cDNA libraries preparedfrom male reproductive tissues of corn as a tool for the identificationof genes expressed in these target tissues, which then facilitates theisolation of 5′ regulatory sequences such as promoters that regulate thegenes.

[0073] Computer-based sequence analyses can be used to identifydifferentially expressed sequences including, but not limited to, thosesequences expressed in one tissue compared with another tissue. Forexample, a different set of sequences can be found from cDNA isolatedfrom root tissue versus leaf tissue. Accordingly, sequences can becompared from cDNA libraries prepared from plants grown under differentenvironmental or physiological conditions. Once the preferred sequencesare identified from the cDNA library of interest, the genomic clones canbe isolated from a genomic library prepared from the plant tissue, andcorresponding regulatory sequences including but not limited to 5′regulatory sequences can be identified and isolated.

[0074] In one preferred embodiment, expressed sequence tags (EST)sequences from a variety of cDNA libraries are catalogued in a sequencedatabase. This database is used to identify promoter targets from aparticular tissue of interest. The selection of expressed sequence tagsfor subsequent promoter isolation is reflective of the presence of oneor more sequences among the representative ESTs from a random samplingof an individual cDNA library or a collection of cDNA libraries. Forexample, the identification of regulatory sequences that direct theexpression of transcripts in male reproductive tissues is conducted byidentifying ESTs found in tissues such as tassel and anther, and absentor in lower abundance in other cDNA libraries in the database. Theidentified EST leads are then evaluated for relative abundance withinthe library and the expression profile for a given EST is assessed. Byabundance as used herein is meant the number of times a clone or clusterof clones appears in a library. The sequences that are enhanced or inhigh abundance in a specific tissue or organ that represent a targetexpression profile are identified in this manner and primers can bedesigned from the identified EST sequences. A PCR-based approach can beused to amplify flanking regions from a genomic library of the targetplant of interest. A number of methods are known to those of skill inthe art to amplify unknown DNA sequences adjacent to a core region ofknown sequence. Methods include but are not limited to inverse PCR(IPCR), vectorette PCR, Y-shaped PCR, and genome walking approaches.

[0075] In a preferred embodiment, genomic DNA ligated to an adaptor issubjected to a primary round of PCR amplification with a gene-specificprimer and a primer that anneals to the adaptor sequence. The PCRproduct is next used as the template for a nested round of PCRamplification with a second gene-specific primer and second adaptor. Theresulting fragments from the nested PCR reaction are then isolated,purified and subcloned into an appropriate vector. The fragments aresequenced, and the translational start sites can be identified when theEST is derived from a truncated cDNA. The fragments can be cloned intoplant expression vectors as transcriptional or translational fusionswith a reporter gene such as β-glucuronidase (GUS). The constructs canbe tested in transient analyses, and subsequently the 5′ regulatoryregions are operably linked to other genes and regulatory sequences ofinterest in a suitable plant transformation vector and the transformedplants are analyzed for the expression of the gene(s) of interest, byany number of methods known to those of skill in the art.

[0076] Any plant can be selected for the identification of genes andregulatory sequences. Examples of suitable plant targets for theisolation of genes and regulatory sequences would include but are notlimited to Acadia, alfalfa, apple, apricot, Arabidopsis, artichoke,arugula, asparagus, avocado, banana, barley, beans, beet, blackberry,blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe,carrot, cassava, castorbean, cauliflower, celery, cherry, chicory,cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton,cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel,figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit,lettuce, leeks, lemon, lily, lime, Loblolly pine, linseed, mango, melon,mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive,onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea,peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radiscchio,radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf,turnip, a vine, watermelon, wheat, yams, and zucchini. Particularlypreferred plant targets would include corn, cotton, rice, soybean, andwheat.

[0077] The nucleic acid molecules of the present invention are isolatedfrom corn (Zea mays). The corn plant develops about 20-21 leaves, silksabout 65 days post-emergence, and matures about 125 days post-emergence.Normal corn plants follow a general pattern of development, but the timeinterval between different stages and morphology varies betweendifferent hybrids, growth and environmental conditions.

[0078] There are a number of identifiable stages in corn plantdevelopment. The stages are defined as vegetative (V) and reproductive(R) stages. Subdivisions of the V stages are numerically designated asV1, V2,V3, etc., through V(n) where (n) represents the last leaf stagebefore tasseling (VT) and the first V stage is the emergence (VE) stage.For example, VE is the emergence from the soil of a seedling leaf, V1represents the first true leaf, V2 represents the second leaf, etc. Thereproductive stages include the first appearance of silk to the matureseed and are represented as follows: R1 is silking, R2 is blistering, R3is the milk stage, R4 is the dough stage, R5 is the dent stage, and R6is physiological maturity (see for example, Ritchie et al. (1986) How aCorn Plant Develops, Iowa State University of Science and TechnologyCooperative Exension Service, Ames, Iowa 48: 1-21).

[0079] Any type of plant tissue can be used as a target tissue for theidentification of genes and associated regulatory sequences. For thepresent invention, corn male reproductive tissue is used. Morepreferably corn tassel tissues are the target tissues for identificationof promoter sequences. Corn cDNA libraries can be constructed fromseveral different plant developmental stages. More preferably cornplants at stages V6-V9 are used. Background or non-target libraries caninclude but are not limited to libraries such as leaf, root, embryo,callus, shoot, seedling, endosperm, culm, ear, and silks.

[0080] Any method that allows a differential comparison betweendifferent types or classes of sequences can be used to isolate genes orregulatory sequences of interest. For example, in one differentialscreening approach, a cDNA library from mRNA isolated from a particulartissue can be prepared in a bacteriophage host using a commerciallyavailable cloning kit. The plaques are spread onto plates containinglawns of a bacterial host such as E. coli to generate bacteriophageplaques. About 10⁵-10⁶ plaques can be lifted onto DNA-binding membranes.Duplicate membranes are probed using probes generated from mRNA from thetarget and non-target or background tissue. The probes are labeled tofacilitate detection after hybridization and development. Plaques thathybridize to target tissue-derived probes but not to non-target tissuederived probes that display a desired differential pattern of expressioncan be selected for further analysis. Genomic DNA libraries can also beprepared from a chosen species by partial digestion with a restrictionenzyme and size selecting the DNA fragments within a particular sizerange. The genomic DNA can be cloned into a suitable vector includingbut not limited to a bacteriophage and prepared using a suitable vectorsuch as a bacteriophage using a suitable cloning kit from any number ofvendors (see for example Stratagene, La Jolla Calif. or Gibco BRL,Gaithersburg, Md.).

[0081] Differential hybridization techniques as described are well knownto those of skill in the art and can be used to isolate a desired classof sequences. By classes of sequences as used herein is meant sequencesthat can be grouped based on a common identifier including but notlimited to sequences isolated from a common target plant, a commonlibrary, or a common plant tissue type. In a preferred embodiment,sequences of interest are identified based on sequence analyses andquerying of a collection of diverse cDNA sequences from libraries ofdifferent tissue types. The disclosed method provides an example of adifferential screening approach based on electronic sequence analyses ofplant ESTs derived from diverse cDNA libraries.

[0082] A number of methods used to assess gene expression are based onmeasuring the mRNA level in an organ, tissue, or cell sample. Typicalmethods include but are not limited to RNA blots, ribonucleaseprotection assays and RT-PCR. In another preferred embodiment, ahigh-throughput method is used whereby regulatory sequences areidentified from a transcript profiling approach. The development of cDNAmicroarray technology enables the systematic monitoring of geneexpression profiles for thousands of genes (Schena et al, Science, 270:467, 1995). This DNA chip-based technology arrays thousands of cDNAsequences on a support surface. These arrays are simultaneouslyhybridized to multiple labeled cDNA probes prepared from RNA samples ofdifferent cell or tissue types, allowing direct comparative analysis ofexpression. This technology was first demonstrated by analyzing 48Arabidopsis genes for differential expression in roots and shoots(Schena et al, Science, 270:467, 1995). More recently, the expressionprofiles of over 1400 genes were monitored using cDNA microarrays (Ruanet al, The Plant Journal 15:821, 1998). Microarrays provide ahigh-throughput, quantitative and reproducible method to analyze geneexpression and characterize gene function. The transcript profilingapproach using microarrays thus provides another valuable tool for theisolation of regulatory sequences such as promoters associated withthose genes.

[0083] The present invention uses high throughput sequence analyses toform the foundation of rapid computer-based identification of sequencesof interest. Those of skill in the art are aware of the resourcesavailable for sequence analyses. Sequence comparisons can be done bydetermining the similarity of the test or query sequence with sequencesin publicly available or proprietary databases (“similarity analysis”)or by searching for certain motifs (“intrinsic sequence analysis”)(e.g., cis elements) (Coulson, Trends in Biotechnology, 12:76, 1994;Birren et al., Genome Analysis, 1:543, 1997).

[0084] The nucleotide sequences provided in SEQ ID NOS: 79-98 orfragments thereof, or complements thereof, or a nucleotide sequence atleast 90% identical, preferably 95% identical even more preferably 99%or 100% identical to the sequence provided in SEQ ID NOS: 79-98 orfragment thereof, or complement thereof, can be “provided” in a varietyof mediums to facilitate use. Such a medium can also provide a subsetthereof in a form that allows one of skill in the art to examine thesequences.

[0085] In one application of this embodiment, a nucleotide sequence ofthe present invention can be recorded on computer readable media. Asused herein, “computer readable media” refers to any medium that can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard disc,storage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. One of skill in theart can readily appreciate how any of the presently known computerreadable media can be used to create a manufacture comprising computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention.

[0086] By providing one or more of nucleotide sequences of the presentinvention, those of skill in the art can routinely access the sequenceinformation for a variety of purposes. Computer software is publiclyavailable that allows one of skill in the art to access sequenceinformation provided in a computer readable medium. Examples of publicdatabases would include but are not limited to the DNA Database of Japan(DDBJ) (http://www.ddbj.nig.ac.jp/);Genbank(http://www.ncbi.nlm.nih.gov/web/Genbank/Index.html); and the EuropeanMolecular Biology Laboratory Nucleic Acid Sequence Database (EMBL)(http://www.ebi.ac.uk/ebi_docs/embl_db.html) or versions thereof. Anumber of different search algorithms have been developed, including butnot limited to the suite of programs referred to as BLAST programs.There are five implementations of BLAST, three designed for nucleotidesequence queries (BLASTN, BLASTX, and TBLASTX) and two designed forprotein sequence queries (BLASTP and TBLASTN) (Coulson, Trends inBiotechnology, 12:76-80, 1994; Birren et al., Genome Analysis, 1:543,1997).

[0087] Any program designed for motif searching also has utility in thepresent invention. Sequence analysis programs designed for motifsearching can be used for identification of cis elements. Preferredcomputer programs would include but are not limited to MEME, SIGNALSCAN, and GENESCAN. MEME is a program that identifies conserved motifs(either nucleic acid or peptide) in a group of unaligned sequences. MEMEsaves these motifs as a set of profiles. These profiles can be used tosearch a database of sequences. A MEME algorithm (version 2.2) can befound in version 10.0 of the GCG package; MEME (Bailey and Elkan,Machine Learning, 21(1-2):51-80,1995 and the location of the website ishttp://www.sdsc.edu/MEME/meme/website/COPYRIGHT.html. SIGNALSCAN is aprogram that identifies known motifs in the test sequences usinginformation from other motif databases (Prestridge, CABIOS 7, 203-206,1991). SIGNALSCAN version 4.0 information is available at the followingwebsite: http://biosci.cbs.umn.edu/software/sigscan.html. The ftp sitefor SIGNALSCAN is ftp://biosci.cbs.umn.edu/software/sigscan.html.Databases used with SIGNALSCAN include PLACE(http://www.dna.affrc.go.ip/htdocs/PLACE; Higo et al., Nucleic AcidsResearch 27(1):297-300, 1999) and TRANSFAC (Heinemeye, X. et al.,Nucleic Acid Research 27(1):318-322) that can be found at the followingwebsite: http://transfac.gbf.de/.GENESCAN is another suitable programfor motif searching (Burge and Karlin, J. Mol. Biol. 268, 78-94, 1997),and version 1.0 information is available at the following website:http://gnomic.stanford.edu/GENESCANW.html. As used herein, “a targetstructural motif” or “target motif” refers any rationally selectedsequence or combination of sequences in which the sequence(s) are chosenbased on a three-dimensional configuration that is formed upon thefolding of the target motif. There are a variety of target motifs knownto those of skill in the art. Protein target motifs include, but are notlimited to, enzymatic active sites and signal sequences. Preferredtarget motifs of the present invention would include but are not limitedto promoter sequences, cis elements, hairpin structures and otherexpression elements such as protein binding sequences.

[0088] As used herein, “search means” refers to one or more programsthat are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means. Search means are used to identifyfragments or regions of the sequences of the present invention thatmatch a particular target sequence or target motif. Multiple sequencescan also be compared in order to identify common regions or motifs thatmay be responsible for specific functions. For example, cis elements orsequence domains that confer a specific expression profile can beidentified when multiple promoter regions of similar classes ofpromoters are aligned and analyzed by certain software packages.

[0089] The present invention further provides systems, particularlycomputer-based systems, that contain the sequence information describedherein. As used herein, a “computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. Those of skill in the art can appreciate thatany one of the available computer-based systems are suitable for use inthe present invention.

[0090] SEQ ID NOS: 4-76 are primers designed from the cDNA sequencesidentified from the computer-based sequence comparisons. These sequencesare used to extend the nucleic acid sequence using polymerase chainreaction (PCR) amplification techniques (see for example, Mullis et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263, 1986; Erlich et al.,European Patent Appln. 50,424; European Patent Appln. 84,796, EuropeanPatent Appln. 258,017, European Patent Appln. 237,362; Mullis, EuropeanPatent Appln. 201,184; Mullis et al., U.S. Pat. No. 4,683,202; Erlich,U.S. Pat. No. 4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194). Anumber of PCR amplification methods are known to those of skill in theart and are used to identify nucleic acid sequences adjacent to a knownsequence. For example, inverse PCR (IPCR) methods to amplify unknown DNAsequences adjacent to a core region of known sequence have beendescribed. Other methods are also available such as capture PCR(Lagerstrom et al., PCR Methods Applic. 1:111, 1991), and walking PCR(Parker et al., Nucleic Acids Res 19:3055, 1991). A number ofmanufacturers have also developed kits based on modifications of thesemethods for the purposes of identifying sequences of interest. Technicaladvances including improvements in primer and adaptor design,improvements in the polymerase enzyme, and thermocycler capabilies havefacilitated quicker, more efficient methods for isolating sequences ofinterest.

[0091] In a preferred embodiment, the flanking sequences containing the5′ regulatory elements of the present invention are isolated using agenome-walking approach (Universal GenomeWalker™ Kit, CLONTECHLaboratories, Inc., Palo Alto, Calif.). In brief, the purified genomicDNA is subjected to a restriction enzyme digest that produces genomicDNA fragments with ends that are ligated with GenomeWalker™ adaptors.GenomeWalker™ primers are used along with gene specific primers in twoconsecutive PCR reactions (primary and nested PCR reactions) to producePCR products containing the 5′ regulatory sequences that aresubsequently cloned and sequenced.

[0092] In addition to their use in modulating gene expression, thepromoter sequences of the present invention also have utility as probesor primers in nucleic acid hybridization experiments. The nucleic acidprobes and primers of the present invention can hybridize understringent conditions to a target DNA sequence. The term “stringenthybridization conditions” is defined as conditions under which a probeor primer hybridizes specifically with a target sequence(s) and not withnon-target sequences, as can be determined empirically. The term“stringent conditions” is functionally defined with regard to thehybridization of a nucleic-acid probe to a target nucleic acid (i.e., toa particular nucleic-acid sequence of interest) by the specifichybridization procedure (see for example Sambrook et al., 1989, at9.52-9.55 and 9.47-9.52, 9.56-9.58; Kanehisa, Nucl. Acids Res.12:203-213, 1984; Wetmur and Davidson, J. Mol. Biol. 31:349-370, 1968).Appropriate stringency conditions that promote DNA hybridization are,for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., and they are known to thoseskilled in the art or can be found in laboratory manuals including butnot limited to Current Protocols in Molecular Biology, John Wiley &Sons, N.Y., 1989, 6.3.1-6.3.6. For example, the salt concentration inthe wash step can be selected from a low stringency of about 2.0×SSC at50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C. Both temperature and salt may be varied, oreither the temperature or the salt concentration may be held constantwhile the other variable is changed. For example, hybridization usingDNA or RNA probes or primers can be performed at 65° C. in 6×SSC, 0.5%SDS, 5× Denhardt's, 100 μg/mL nonspecific DNA (e.g., sonicated salmonsperm DNA) with washing at 0.5×SSC, 0.5% SDS at 65° C., for highstringency.

[0093] It is contemplated that lower stringency hybridization conditionssuch as lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA fragments. Detection ofDNA segments via hybridization is well-known to those of skill in theart. Thus depending on the application envisioned, one will desire toemploy varying hybridization conditions to achieve varying degrees ofselectivity of probe towards target sequence and the method of choicewill depend on the desired results.

[0094] The nucleic acid sequences in SEQ ID NOS: 79-98, and any variantsthereof, are capable of hybridizing to other nucleic acid sequencesunder appropriately selected conditions of stringency. As used herein,two nucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibit complementarity. As used herein, molecules are said toexhibit “complete complementarity” when every nucleotide of one of themolecules is complementary to a nucleotide of the other. Two moleculesare said to be “minimally complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under at least conventional “low stringency” conditions.Similarly, the molecules are said to be “complementary” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under conventional “high stringency”conditions. Conventional stringency conditions are described by Sambrooket al. (Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989), and by Haymes et al.(Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C., 1985).

[0095] In a preferred embodiment, the nucleic acid sequences SEQ ID NOS:79-98 or a fragment, region, cis element, or oligomer of these sequencesmay be used in hybridization assays of other plant tissues to identifyclosely related or homologous genes and associated regulatory sequences.These include but are not limited to Southern or northern hybridizationassays on any substrate including but not limited to an appropriatelyprepared plant tissue, cellulose, nylon, or combination filter, chip, orglass slide. Such methodologies are well known in the art and areavailable in a kit or preparation that can be supplied by commercialvendors.

[0096] Of course, nucleic acid fragments can also be obtained by othertechniques such as by directly synthesizing the fragment by chemicalmeans, as is commonly practiced by using an automated oligonucleotidesynthesizer. Fragments can also be obtained by application of nucleicacid reproduction technology, such as the PCR™ (polymerase chainreaction) technology or by recombinant DNA techniques generally known tothose of skill in the art of molecular biology. Regarding theamplification of a target nucleic-acid sequence (e.g., by PCR) using aparticular amplification primer pair, “stringent PCR conditions” referto conditions that permit the primer pair to hybridize only to thetarget nucleic-acid sequence to which a primer having the correspondingwild-type sequence (or its complement) would bind and preferably toproduce a unique amplification product.

[0097] A fragment of a nucleic acid as used herein is a portion of thenucleic acid that is less than full-length. For example, for the presentinvention any length of nucleotide sequence that is less than thedisclosed nucleotide sequences of SEQ ID NOS: 79-98 is considered to bea fragment. A fragment can also comprise at least a minimum lengthcapable of hybridizing specifically with a native nucleic acid understringent hybridization conditions as defined above. The length of sucha minimal fragment is preferably at least 8 nucleotides, more preferably15 nucleotides, even more preferably at least 20 nucleotides, and mostpreferably at least 30 nucleotides of a native nucleic acid sequence.

[0098] The nucleic acid sequences of the present invention can also beused as probes and primers. Nucleic acid probes and primers can beprepared based on a native gene sequence. A “probe” is an isolatednucleic acid to which is attached a conventional detectable label orreporter molecule, e.g., a radioactive isotope, ligand, chemiluminescentagent, or enzyme. “Primers” are isolated nucleic acids that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs can be used for amplification of a nucleic acidsequence, e.g., by the polymerase chain reaction (PCR) or otherconventional nucleic-acid amplification methods.

[0099] Probes and primers are generally 15 nucleotides or more inlength, preferably 20 nucleotides or more, more preferably 25nucleotides, and most preferably 30 nucleotides or more. Such probes andprimers hybridize specifically to a target DNA or RNA sequence underhigh stringency hybridization conditions and hybridize specifically to atarget native sequence of another species under lower stringencyconditions. Preferably, probes and primers according to the presentinvention have complete sequence similarity with the native sequence,although probes differing from the native sequence and that retain theability to hybridize to target native sequences may be designed byconventional methods. Methods for preparing and using probes and primersare described (see Sambrook et al., 1989; Ausubel et al., 1992, andInnis et al., 1990). PCR-primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer (Version 0.5, © 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass.). Primers and probes based on thenative promoter sequences disclosed herein can be used to confirm and,if necessary, to modify the disclosed sequences by conventional methods,e.g., by re-cloning and re-sequencing.

[0100] In another embodiment, the nucleotide sequences of the promotersdisclosed herein can be modified. Those skilled in the art can createDNA molecules that have variations in the nucleotide sequence. Thenucleotide sequences of the present invention as shown in SEQ ID NOS:79-98 may be modified or altered to enhance their controlcharacteristics. One preferred method of alteration of a nucleic acidsequence is to use PCR to modify selected nucleotides or regions ofsequences. These methods are known to those of skill in the art.Sequences can be modified, for example by insertion, deletion orreplacement of template sequences in a PCR-based DNA modificationapproach. “Variant” DNA molecules are DNA molecules containing changesin which one or more nucleotides of a native sequence is deleted, added,and/or substituted, preferably while substantially maintaining promoterfunction. In the case of a promoter fragment, “variant” DNA can includechanges affecting the transcription of a minimal promoter to which it isoperably linked. Variant DNA molecules can be produced, for example, bystandard DNA mutagenesis techniques or by chemically synthesizing thevariant DNA molecule or a portion thereof.

[0101] In another embodiment, the nucleotide sequences as shown in SEQID NOS: 79-98 include any length of said nucleotide sequences that arecapable of regulating an operably linked DNA sequence. For example, thesequences as disclosed in SEQ ID NOS: 79-98 may be truncated or portionsdeleted and still be capable of regulating transcription of an operablylinked DNA sequence. In a related embodiment, a cis element of thedisclosed sequences may confer a particular specificity such asconferring enhanced expression of operably linked DNA sequences incertain tissues. Consequently, any sequence fragments, portions, orregions of the disclosed sequences of SEQ ID NOS: 79-98 can be used asregulatory sequences including but not limited to cis elements or motifsof the disclosed sequences. For example, one or more base pairs may bedeleted from the 5′ or 3′ end of a promoter sequence to produce a“truncated” promoter. One or more base pairs can also be inserted,deleted, or substituted internally to a promoter sequence. Promoters canbe constructed such that promoter fragments or elements are operablylinked for example, by placing such a fragment upstream of a minimalpromoter. A minimal or basal promoter is a piece of DNA that is capableof recruiting and binding the basal transcription machinery. One exampleof basal transcription machinery in eukaryotic cells is the RNApolymerase II complex and its accessory proteins. The enzymaticcomponents of the basal transcription machinery are capable ofinitiating and elongating transcription of a given gene, utilizing aminimal or basal promoter. That is, there are not added cis-actingsequences in the promoter region that are capable of recruiting andbinding transcription factors that modulate transcription, e.g.,enhance, repress, render transcription hormone-dependent, etc.Substitutions, deletions, insertions or any combination thereof can becombined to produce a final construct.

[0102] Native or synthetic nucleic acids according to the presentinvention can be incorporated into recombinant nucleic acid constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. In one preferred embodiment, the nucleotide sequences ofthe present invention as shown in SEQ ID NOS: 79-98 or fragments,variants or derivatives thereof are incorporated into an expressionvector cassette that includes the promoter regions of the presentinvention operably linked to a genetic component such as a selectable,screenable, or scorable marker gene. The disclosed nucleic acidsequences of the present invention are preferably operably linked to agenetic component such as a nucleic acid that confers a desirablecharacteristic associated with plant morphology, physiology, growth anddevelopment, yield, nutritional enhancement, disease or pest resistance,or environmental or chemical tolerance. These genetic components such asmarker genes or agronomic genes of interest can function in theidentification of a transformed plant cell or plant, or a produce aproduct of agronomic utility.

[0103] In a preferred embodiment, one genetic component produces aproduct that serves as a selection device and functions in a regenerableplant tissue to produce a compound that would confer upon the planttissue resistance to an otherwise toxic compound. Genes of interest foruse as a selectable, screenable, or scorable marker would include butare not limited to GUS (coding sequence for beta-glucuronidase), GFP(coding sequence for green fluorescent protein), LUX (coding gene forluciferase), antibiotic resistance marker genes, or herbicide tolerancegenes. Examples of transposons and associated antibiotic resistancegenes include the transposons Tns (bla), Tn5 (nptII), Tn7 (dhfr),penicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate(and trimethoprim); chloramphenicol; and tetracycline.

[0104] Characteristics useful for selectable markers in plants have beenoutlined in a report on the use of microorganisms (Advisory Committee onNovel Foods and Processes, July 1994). These include stringent selectionwith minimum number of nontransformed tissues, large numbers ofindependent transformation events with no significant interference withthe regeneration, application to a large number of species, andavailability of an assay to score the tissues for presence of themarker.

[0105] A number of selectable marker genes are known in the art andseveral antibiotic resistance markers satisfy these criteria, includingthose resistant to kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4). Useful dominant selectable marker genesinclude genes encoding antibiotic resistance genes (e.g., resistance tohygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin);and herbicide resistance genes (e.g., phosphinothricinacetyltransferase). A useful strategy for selection of transformants forherbicide resistance is described, e.g., in Vasil (Cell Culture andSomatic Cell Genetics of Plants, Vols. I-III, Laboratory Procedures andTheir Applications Academic Press, New York, 1984). Particularlypreferred selectable marker genes for use in the present invention wouldinclude genes that confer resistance to compounds such as antibioticslike kanamycin and herbicides like glyphosate (Della-Cioppa et al.,Bio/Technology 5(6), 1987; U.S. Pat. No. 5,463,175; U.S. Pat. No.5,633,435). Other selection devices can also be implemented and wouldstill fall within the scope of the present invention.

[0106] For the practice of the present invention, conventionalcompositions and methods for preparing and using vectors and host cellsare employed, as discussed, inter alia, in Sambrook et al., 1989. In apreferred embodiment, the host cell is a plant cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.(Cloning Vectors: A Laboratory Manual, 1985, supp. 1987); Weissbach andWeissbach (Methods for Plant Molecular Biology, Academic Press, 1989);Gelvin et al. (Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990); and Croy (Plant Molecular Biology LabFax, BIOSScientific Publishers, 1993). Plant expression vectors can include, forexample, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences. They can also include aselectable marker as described to select for host cells containing theexpression vector. Such plant expression vectors also contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally or developmentally regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and a polyadenylation signal. Other sequences ofbacterial origin are also included to allow the vector to be cloned in abacterial host. The vector will also typically contain a broad hostrange prokaryotic origin of replication. In a particularly preferredembodiment, the host cell is a plant cell and the plant expressionvector comprises a promoter region as disclosed in SEQ ID NOS: 79-98, anoperably linked transcribable sequence, and a transcription terminationsequence. Other regulatory sequences envisioned as genetic components inan expression vector include, but is not limited to, non-translatedleader sequence that can be coupled with the promoter. Plant expressionvectors also can comprise additional sequences including but not limitedto restriction enzyme sites that are useful for cloning purposes.

[0107] A number of promoters have utility for plant gene expression forany gene of interest including but not limited to selectable markers,scorable markers, genes for pest tolerance, disease tolerance,nutritional enhancements and any other gene that confers a desirabletrait or characteristic. Examples of constitutive promoters useful forplant gene expression include, but are not limited to, the cauliflowermosaic virus (CaMV) 35S promoter, which confers constitutive, high-levelexpression in most plant tissues (see, e.g., Odel et al., Nature313:810, 1985), including monocots (see, e.g., Dekeyser et al., PlantCell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389, 1990);the nopaline synthase promoter (An et al., Plant Physiol. 88:547, 1988);the octopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989);and the figwort mosaic virus (FMV) promoter as described in U.S. Pat.No. 5,378,619.

[0108] A variety of plant gene promoters that are regulated in responseto environmental, hormonal, chemical, and/or developmental signals canbe used for expression of an operably linked gene in plant cells,including promoters regulated by (1) heat (Callis et al., Plant Physiol.88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al.,Plant Cell 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, PlantCell 3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpsonet al., EMBO J. 4:2723, 1985), (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell 1:969, 1989), (4) wounding (e.g., wunI,Siebertz et al., Plant Cell 1:961, 1989); or (5) chemicals such asmethyl jasmonate, salicylic acid, or safener. It may also beadvantageous to employ (6) organ-specific promoters (e.g., Roshal etal., EMBO J. 6:1155, 1987; Schernthaner et al., EMBO J. 7:1249, 1988;Bustos et al., Plant Cell 1:839, 1989). The promoters of the presentinvention are plant promoters that are capable of transcribing operablylinked DNA sequences in male reproductive tissues and can be operablylinked to any gene of interest in an expression vector.

[0109] Plant expression vectors can include RNA processing signals,e.g., introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. In addition, theexpression vectors may include additional regulatory sequences from the3′-untranslated region of plant genes (Thornburg et al., Proc. Natl.Acad. Sci. USA 84:744, 1987; An et al., Plant Cell 1:115, 1989), e.g., a3′ terminator region to increase mRNA stability of the mRNA, such as thePI-II terminator region of potato or the octopine or nopaline synthase3′ terminator regions. Five-end non-translated regions of a mRNA canplay an important role in translation initiation and can also be agenetic component in a plant expression vector. For example,non-translated 5′ leader sequences derived from heat shock protein geneshave been demonstrated to enhance gene expression in plants (see, forexample U.S. Pat. No. 5,362,865). These additional upstream anddownstream regulatory sequences may be derived from a source that isnative or heterologous with respect to the other elements present on theexpression vector.

[0110] The promoter sequences of the present invention are used tocontrol gene expression in plant cells. The disclosed promoter sequencesare genetic components that are part of vectors used in planttransformation. The promoter sequences of the present invention can beused with any suitable plant transformation plasmid or vector containinga selectable or screenable marker and associated regulatory elements, asdescribed, along with one or more nucleic acids expressed in a mannersufficient to confer a particular desirable trait. Examples of suitablestructural genes of agronomic interest envisioned by the presentinvention would include but are not limited to one or more genes forinsect tolerance, such as a B.t., pest tolerance such as genes forfungal disease control, herbicide tolerance such as genes conferringglyphosate tolerance, and genes for quality improvements such as yield,nutritional enhancements, environmental or stress tolerances, or anydesirable changes in plant physiology, fertilizer, growth, development,morphology or plant product(s).

[0111] Alternatively, the DNA coding sequences can effect thesephenotypes by encoding a non-translatable RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev. 9:207,1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillitoe, Mol.Biotech. 7:125,1997). Thus, any gene that produces a protein or mRNAthat expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

[0112] In addition to regulatory elements or sequences located upstream(5′) or within a DNA sequence, there are downstream (3′) sequences thataffect gene expression and thus the term regulatory sequence as usedherein refers to any nucleotide sequence located upstream, within, ordownstream to a DNA sequence that controls, mediates, or affectsexpression of a gene product in conjunction with the protein syntheticapparatus of the cell.

[0113] The promoter sequences of the present invention may be modified,for example for expression in other plant systems. In another approach,novel hybrid promoters can be designed or engineered by a number ofmethods. Many promoters contain upstream sequences that activate,enhance or define the strength and/or specificity of the promoter(Atchison, Ann. Rev. Cell Biol. 4:127, 1988). T-DNA genes, for example,contain “TATA” boxes defining the site of transcription initiation andother upstream elements located upstream of the transcription initiationsite modulate transcription levels (Gelvin, In: Transgenic Plants, Kungand Us, eds, San Diego: Academic Press, pp.49-87, 1988). Ni andcolleagues combined a trimer of the octopine synthase (ocs) activator tothe mannopine synthase (mas) activator plus promoter and reported anincrease in expression of a reporter gene (Ni et al., The Plant Journal7:661, 1995). The upstream regulatory sequences of the present inventioncan be used for the construction of such chimeric or hybrid promoters.Methods for construction of variant promoters of the present inventioninclude but are not limited to combining control elements of differentpromoters or duplicating portions or regions of a promoter (see forexample U.S. Pat. No. 5,110,732 and U.S. Pat. No. 5,097,025). Those ofskill in the art are familiar with the standard resource materials thatdescribe specific conditions and procedures for the construction,manipulation and isolation of macromolecules (e.g., DNA molecules,plasmids, etc.), generation of recombinant organisms and the screeningand isolation of genes, (see for example Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; Maliga etal., Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997), volume2, Detecting Genes, (1998), volume 3, Cloning Systems, (1999) volume 4,Mapping Genomes, (1999), Cold Spring Harbor, N.Y.).

[0114] The promoter sequences of the present invention may beincorporated into an expression vector using screenable or scorablemarkers as described and tested in transient analyses that provide anindication of gene expression in stable plant systems. Methods oftesting gene expression in transient assays are known to those of skillin the art. Transient expression of marker genes has been reported usinga variety of plants, tissues and DNA delivery systems. For example,types of transient analyses can include but are not limited to directgene delivery via electroporation or particle bombardment of tissues inany transient plant assay using any plant species of interest. Suchtransient systems would include but are not limited to protoplasts fromsuspension cultures in wheat (Zhou et al., Plant Cell Reports 12:612.1993, electroporation of leaf protoplasts of wheat (Sethi et al., J.Crop Sci. 52: 152, 1983; electroporation of protoplast prepared fromcorn tissue (Sheen, The Plant Cell 3: 225, 1991), or particlebombardment of specific tissues of interest. The present inventionencompasses the use of any transient expression system to evaluateregulatory sequences operatively linked to selected reporter genes,marker genes or agronomic genes of interest. Examples of plant tissuesenvisioned to test in transients via an appropriate delivery systemwould include, but are not limited to, leaf base tissues, callus,cotyledons, roots, endosperm, embryos, floral tissue, pollen, andepidermal tissue.

[0115] Any scorable or screenable marker can be used in a transientassay. Preferred marker genes for transient analyses of the promoters or5′ regulatory sequences of the present invention include a GUS gene or aGFP gene. The expression vectors containing the 5′ regulatory sequencesoperably linked to a marker gene are delivered to the tissues and thetissues are analyzed by the appropriate mechanism, depending on themarker. The quantitative or qualitative analyses are used as a tool toevaluate the potential expression profile of the 5′ regulatory sequenceswhen operatively linked to genes of agronomic interest in stable plants.Ultimately, the 5′ regulatory sequences of the present invention aredirectly incorporated into suitable plant transformation expressionvectors comprising the 5′ regulatory sequences operatively linked to atranscribable DNA sequence interest, transformed into plants and thestably transformed plants and progeny thereof analyzed for the desiredexpression profile conferred by the 5′ regulatory sequences.

[0116] Those of skill in the art are aware of the vectors suitable forplant transformation. Suitable vectors would include but are not limitedto disarmed Ti-plasmids for Agrobacterium-mediated methods. Thesevectors can contain a resistance marker, 1-2 T-DNA borders, and originsof replication for E. coli and Agrobacterium along with one or moregenes of interest and associated regulatory regions. Those of skill inthe art are aware that for Agrobacterium-mediated approaches a number ofstrains and methods are available. Such strains would include but arenot limited to Agrobacterium strains C58, LBA4404, EHA101 and EHA105.Particularly preferred strains are Agrobacterium tumefaciens strains.Other DNA delivery systems for plant transformation are also known tothose of skill in the art and include, but are not limited to, particlebombardment of selected plant tissues.

[0117] Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term exogenous is alsointended to refer to genes that are not normally present in the cellbeing transformed, or perhaps simply not present in the form, structure,etc., as found in the transforming DNA segment or gene, or genes thatare normally present yet which one desires, e.g., to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

[0118] The plant transformation vectors containing the promotersequences of the present invention may be introduced into plants by anyplant transformation method. Several methods are available forintroducing DNA sequences into plant cells and are well known in theart. Suitable methods include but are not limited to bacterialinfection, binary bacterial artificial chromosome vectors, directdelivery of DNA (e.g. via PEG-mediated transformation,desiccation/inhibition-mediated DNA uptake, electroporation, agitationwith silicon carbide fibers), and acceleration of DNA coated particles(reviewed in Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol., 42:205, 1991).

[0119] Methods for specifically transforming dicots primarily useAgrobacterium tumefaciens. For example, transgenic plants reportedinclude but are not limited to cotton (U.S. Pat. No. 5,004,863; U.S.Pat. No. 5,159,135; U.S. Pat. No. 5,518,908, WO 97/43430), soybean (U.S.Pat. No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe et al.,Bio/Technology, 6:923, 1988; Christou et al., Plant Physiol., 87:671,1988); Brassica (U.S. Pat. No. 5,463,174), and peanut (Cheng et al.,Plant Cell Rep., 15: 653, 1996).

[0120] Similar methods have been reported in the transformation ofmonocots. Transformation and plant regeneration using these methods havebeen described for a number of crops including but not limited toasparagus (Asparagus officinalis; Bytebier et al., Proc. Natl. Acad.Sci. U.S.A., 84: 5345, 1987); barley (Hordeum vulgarae; Wan and Lemaux,Plant Physiol., 104: 37, 1994); maize (Zea mays; Rhodes et al., Science,240: 204, 1988; Gordon-Kamm et al., Plant Cell, 2: 603, 1990; Fromm etal., Bio/Technology, 8: 833, 1990; Koziel et al., Bio/Technology, 11:194, 1993); oats (Avena sativa; Somers et al., Bio/Technology, 10: 1589,1992); orchardgrass (Dactylis glomerata; Horn et al., Plant Cell Rep.,7: 469, 1988); rice (Oryza sativa, including indica and japonicavarieties, Toriyama et al., Bio/Technology, 6: 10, 1988; Zhang et al.,Plant Cell Rep., 7: 379, 1988; Luo and Wu, Plant Mol. Biol. Rep., 6:165, 1988; Zhang and Wu, Theor. Appl. Genet., 76: 835, 1988; Christou etal., Bio/Technology, 9: 957, 1991); sorghum (Sorghum bicolor; Casas etal., Proc. Natl. Acad. Sci. U.S.A., 90: 11212, 1993); sugar cane(Saccharum spp.; Bower and Birch, Plant J., 2: 409, 1992); tall fescue(Festuca arundinacea; Wang et al., Bio/Technology, 10: 691, 1992);turfgrass (Agrostis palustris; Zhong et al., Plant Cell Rep., 13: 1,1993); wheat (Triticum aestivum; Vasil et al., Bio/Technology, 10: 667,1992; Weeks et al., Plant Physiol., 102: 1077, 1993; Becker et al.,Plant, J. 5: 299, 1994), and alfalfa (Masoud et al., Transgen. Res., 5:313, 1996). It is apparent to those of skill in the art that a number oftransformation methodologies can be used and modified for production ofstable transgenic plants from any number of target crops of interest.

[0121] The transformed plants are analyzed for the presence of the genesof interest and the expression level and/or profile conferred by thepromoter sequences of the present invention. Those of skill in the artare aware of the numerous methods available for the analysis oftransformed plants. A variety of methods are used to assess geneexpression and determine if the introduced gene(s) is integrated,functioning properly, and inherited as expected. For the presentinvention the promoters can be evaluated by determining the expressionlevels of genes to which the promoters are operatively linked. Apreliminary assessment of promoter function can be determined by atransient assay method using reporter genes, but a more definitivepromoter assessment can be determined from the analysis of stableplants. Methods for plant analysis include but are not limited toSouthern blots or northern blots, PCR-based approaches, biochemicalanalyses, phenotypic screening methods, field evaluations, andimmunodiagnostic assays.

[0122] The methods of the present invention including but not limited tocDNA library preparation, genomic library preparation, sequencing,sequence analyses, PCR technologies, vector construction, transientassays, and plant transformation methods are well known to those ofskill in the art and are carried out using standard techniques ormodifications thereof.

[0123] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention, therefore all matter set forth or shown in theaccompanying drawings is to be interpreted as illustrative and not in alimiting sense.

EXAMPLES Example 1

[0124] Plant Material, DNA Isolation and Library Construction

[0125] The target cDNA libraries included three tassel libraries. Thebackground cDNA libraries included cDNA libraries prepared from leaf,root, embryo, callus, shoot, seedling, endosperm, culm, ear, and silks.

[0126] Plant Growth Conditions

[0127] Seeds are planted at a depth of about 3 cm in soil into 2″-3″pots containing Metro 200 growing medium and transplanted into larger10″ pots containing the same soil after 2-3 weeks. Plants werefertilized as needed. A total of about 900 mg Fe is added to each pot.Corn plants are grown in the greenhouse in 15 hr day/9 hr night cycles.The daytime temperature is 26.7° C. and the night temperature is 21.1°C. Lighting is provided by 1000 W sodium vapor lamps.

[0128] Tissue Isolation

[0129] The corn immature tassel cDNA library (SATMON001) is generatedusing maize (B73, Illinois Foundation Seeds, Champaign, Ill. U.S.A.).The corn plant is at the V6 plant developmental stage. The tassel is animmature tassel 2-3 cm in length. The immature tassel is frozen inliquid nitrogen and the harvested tissue is stored at −80° C. until theRNA is prepared.

[0130] The corn immature tassel cDNA library (SATMON021) is generatedusing maize (DK604, Dekalb Genetics, Dekalb Ill., U.S.A.). The cornplant is at the V8 plant developmental stage. The tassels, which areabout 15-20 cm in length, are collected and frozen in liquid nitrogen.The harvested tissue is stored at −80° C. until the RNA is prepared.

[0131] The corn immature tassel cDNA library (SATMON024) containingtassel with glume, anthers, and pollen is generated using maize (DK604,Dekalb Genetics, Dekalb, Ill. U.S.A.). The corn plant is at the V9 plantdevelopmental stage. The tassel is at the rapidly developing stage, anda tassel about 37 cm along with the glume, anthers, and pollen arecollected and frozen in liquid nitrogen. The harvested tissue is storedat −80° C. until the RNA is prepared.

[0132] The RNA is purified using Trizol reagent available from LifeTechnologies (Gaithersburg, Md.) essentially as recommended by themanufacturer. Poly A+ RNA (mRNA) is purified using magnetic oligo dTbeads essentially as recommended by the manufacturer (Dynabeads, DynalCorporation, Lake Success, N.Y.). Two modifications to the Trizolprotocol include centrifuging the ground tissue samples at 12,000×g for10 minutes at 4° C. after the addition of Trizol to remove insolublematerial, and precipitating the RNA with 0.25 mL isopropanol and 0.25 mL0.8M NaCl per 1.0 mL Trizol used. All the samples are precipitated with0.1 volume of 3M NaOAc and 3.0 volumes of ethanol. RNAs are resuspendedin distilled water at a concentration of 2 μg/μL. The RNAs are DNasetreated for 10 minutes at room temperature with RNase free DNase (BMB,Indianapolis, Ind.) and samples are extracted with phenol/chloroform andisopropanol precipitated as described, and resuspended in distilledwater at a concentration of 1 μg/μL.

[0133] Construction of cDNA libraries is well-known in the art and anumber of cloning strategies exist. A number of cDNA libraryconstruction kits are commercially available. The Superscript™ PlasmidSystem for cDNA synthesis and Plasmid Cloning (Gibco BRL, LifeTechnologies, Gaithersburg, Md.) is used, following the conditionssuggested by the manufacturer.

[0134] The cDNA libraries are plated on LB agar containing theappropriate antibiotics for selection and incubated at 37° C. for asufficient time to allow the growth of individual colonies. Singlecolonies are individually placed in each well of a 96-well microtiterplate containing LB liquid including selective antibiotics. The platesare incubated overnight at approximately 37° C. with gentle shaking topromote growth of the cultures. The plasmid DNA is isolated from eachclone using Qiaprep Plasmid Isolation kits, using the conditionsrecommended by the manufacturer (Qiagen Inc., Santa Clara, Calif.).

[0135] Template plasmid DNA clones are used for subsequent sequencing.For sequencing, the ABI PRISM dRhodamine Terminator Cycle SequencingReady Reaction Kit with AmpliTaq® DNA Polymerase, FS, is used (PEApplied Biosystems, Foster City, Calif.).

[0136] After cDNA synthesis, the samples are diluted with one volume ofwater, and one microliter is used for each tissue-specificity PCR assay.Corn cDNAs were synthesized for tissue specificity testing methods wellknown in the art include leaf, root, rachus, early anther, kernals from6 cm ear, kernals from 4 cm ears, silk, glumme/lemma/palea, matureanther and pollen, meristem, microspores, culm, tassel, ear, and husk.

Example 2

[0137] Promoter Lead Identification

[0138] The database of EST sequences derived from the cDNA librariesprepared from various corn tissues is used to identify the genes withthe correct expression profile from which promoter candidates can beisolated for expression of operably linked DNA sequences in malereproductive tissues. The sequences are also used as query sequencesagainst GenBank databases that contain previously identified andannotated sequences and searched for regions of homology using BLASTprograms. The selection of expressed sequence tags (ESTs) for subsequentpromoter isolation is reflective of the presence of one or moresequences among the representative ESTs from a random sampling of anindividual cDNA library or collection of cDNA libraries. To identifyregulatory sequences that regulate the expression of transcripts in thetarget tissues of interest from EST sequences in the database, asubsetting function is done, requesting ESTs found in target librariessuch as the three tassel libraries and EST clones in all other librarieswere subtracted. The background or non-target libraries included thefollowing: SATMON013 (corn meristem), SATMON020 (corn callus), SATMON022(corn immature ear), SATMON023 (corn ear, growing silks), SATMON025(corn regenerating callus), SATMON004 (corn leaf), SATMON009 (corn leaf,V8 stage), SATMON011 (corn leaf, undeveloped), SATMON016 (corn sheath),SATMON026 (corn leaf), SATMON027 (corn leaf, water stressed 6 days),SATMON031 (corn leaf V4 stage), SATMONN01 (corn leaf normalize),SATMON003 (corn root), SATMON007 (corn primary root), SATMON010 (cornroot V8 stage), SATMON028 (corn root, water stressed 6 days), SATMON030(corn root V4 stage), SATMONN05 (corn root, normalize), SATMON014 (cornendosperm, 14 days after pollination), SATMON017 (corn embryo, 21 daysafter pollination), SATMON033 (corn embryo, 13 days after pollination),SATMON036 (corn endosperm 22 days after pollination), SATMONN04 (cornembryo, 21-DAP, normalized), SATMONN06 (corn embryo, 21-DAP,normalized), SATMON008 (corn primary shoot), SATMON012 (corn seedling, 2days post-germination), SATMON019 (corn culm), SATMON029 (corn seedling,etiolated 4 days), and SATMON034 (corn seedling, cold stressed). ThecDNA clones identified from the subsetting are candidates fortissue-enhanced/specific genes and are further pursued. Rt-PCR reactionsare performed for each of the leads to determine if amale-specific/enhanced pattern of expression is observed. The ESTsequences that were detectable predominantly in male tissues are used toisolate the tissue enhanced/specific promoters.

[0139] Table 1 provides background clone ID (EST) information andGenBank identifier (gi) information for the ESTs used for subsequentisolation of the promoter sequences of SEQ ID NOS: 79-98. The promoterleads were all obtained from the SATMON024 library source as describedabove. Sequence annotation is listed for the clone IDs based on aGenBank BLAST search with a p-value cut-off of 10⁻⁸. The information issubject to change as new sequences are submitted to the sequencedatabases. The annotations for the ESTs are listed as follows with theannotation information in parentheses: Clone ID 700352826); Clone ID700353038 (Cynodon dactyloncalcium-binding pollen allergen gene, partialcds; p value 7e-53); Clone ID 700354918 (Phleum pratense mRNA forpolygalacturonase, partial: p-value 1e-08); Clone ID 700353844 (Z. mayspollen specific mRNA C-terminal (clone 4H7) p-value 6e-26); Clone ID700355306 (Holcus lanatus mRNA for major group I allergen Hol 1 1;p-value le-21)); Clone ID 700353142); Clone ID 700282503 ); Clone ID700282409 (Z. mays ZmPRO1 mRNA for profilin 1; p value 2e-34); Clone ID700352616); Clone ID 700354681 (Z. mays ZmPRO2 mRNA for profilin 2;p-value e-137); 700353007); 700352625); and Clone ID 700382630 (Z. maysmRNA for pectin methylesterase-like protein; p value 1e-08). TABLE 1Promoter Summary Information Clone ID SEQ ID NO. GenBank Identifier (gi)700352826 79 none 700353038 80, 81 g1864023 700354918 82 aj238848700353844 83 x57275 700355306 84 aj012714 700353142 85 none 700282503 86none 700282409 87 x73279 700282409 88 x73279 700352616 89 none 70035468190 x73280 700353007 91 none 700352625 92 none 700382630 93 y13285700352826 94 none 700353038 95, 96, 97, 98 g1864023

Example 3

[0140] Genomic Library Construction, PCR Amplification and PromoterIsolation

[0141] A number of methods are known to those of skill in the art forgenomic library preparation. For genomic libraries of the presentinvention, corn DNA (Maize hybrid Fr27×FrMol17) is isolated by a CsClpurification protocol according to Ausubel et al. (1992) or by a CTABpurification method (Rogers and Bendich, Plant Mol. Biol., 5:69, 1988).Reagents are available commercially (see, for example Sigma ChemicalCo., St. Louis, Mo.). The libraries are prepared according tomanufacturer instructions (GENOME WALKER, a trademark of CLONTECHLaboratories, Inc, Palo Alto, Calif.). In separate reactions, genomicDNA is subjected to restriction enzyme digestion overnight at 37° C.with the following blunt-end endonucleases: EcoRV, ScaI, DraI, PvuII, orStuI (CLONTECH Laboratories, Inc., Palo Alto, Calif.). The reactionmixtures are extracted with phenol:chloroform, ethanol precipitated, andresuspended in Tris-EDTA buffer. The purified blunt-ended genomic DNAfragments are then ligated to the GenomeWalker™ adaptors and ligation ofthe resulting DNA fragments to adaptors were done according tomanufacturer protocol. The GenomeWalker™ sublibraries are aliquoted andstored at −20° C.

[0142] Genomic DNA ligated to the GenomeWalker™ adaptor (above) issubjected to a primary round of PCR amplification with gene-specificprimer 1 (GSP1) and a primer that anneals to the Adaptor sequence,adaptor primer 1 (AP1) (SEQ ID NO:1). A diluted (1:50) aliquot of theprimary PCR reaction is used as the input DNA for a nested round of PCRamplification with gene-specific primer 2 (GSP2) and adaptor primer 2(AP2) (SEQ ID NO:2), or adaptor primer 3 (AP3) (SEQ ID NO:3). Theannealing temperatures of the GenomeWalker™ primary primer (AP1) andnested primer (AP2) are 59° C. and 71° C., respectively. Generally, genespecific primers are designed to have the following characteristics:26-30 nucleotides in length, GC content of 40-60% with resultingtemperatures for most of the gene specific primers in the high 60° C.range or about 70° C. The Taq polymerase used is Amplitaq Gold™ orExpand HiFidelity (Boehringer Mannheim) available through Perkin-ElmerBiosystems (Branchbury, N.J.). A number of temperature cyclinginstruments and reagent kits are commercially available for performingPCR experiments and include those available from PE Biosystems (FosterCity, Calif.), Stratagene (La Jolla, Calif.), and MJ Research Inc.(Watertown, Mass.). Following the primary PCR reaction, an aliquot istaken (10-15 μL) for agarose gel analysis. Isolation of each unknownsequence required amplification from 5 sub-genomic libraries and anegative control (without DNA).

[0143] The PCR components and conditions generally used are outlinedbelow: PRIMARY PCR Component Amount/Volume required Sub-library aliquot1 μL Gene-specific primer 1 1 μL (100 pmol) GenomeWalker ™ Adaptorprimer 1 (AP1) 1 μL dNTP mix (10 mM of each dNTP) 1 μL DMSO 2.5 μL (or2-5% final concentration) 10X PCR buffer (containing MgCl₂) 5 μL (finalconcentration of 1X) Amplitaq Gold ™ or Expand HiFidelity 0.5 μLDistilled Water For final reaction volume of 50 μL Reaction Conditionsfor Primary PCR: A. 1 minutes at 95° C. B. 94° C. for 2 seconds, 70° C.for 3 minutes; repeat 94° C./70° C. cycling for total of 7 times C. 94°C. for 2 seconds, 65° C. for 3 minutes; repeat 94° C./65° C. cycling fortotal of 36 times D. 65° C. for 4 minutes as a final extension E. 10° C.for an extended incubation NESTED PCR (secondary PCR reaction) ComponentAmount/Volume Required 1:50 dilution of the primary PCR reaction 1 μLGene-specific primer 2 1 μL (100 pmol) GenomeWalker ™ Adaptor primer 2or 3 1 μL (AP2 or AP3)) dNTP mix (10 mM of each dNTP) 1 μL DMSO 2.5 μL10X PCR buffer (containing MgCl₂) 5 μL (final concentration of 1X)Amplitaq Gold ™ 0.5 μL Distilled water to final reaction volume of 50 μLReaction Conditions for Nested PCR: A. 1 minutes at 95° C. B. 94° C. for2 seconds, 70° C. for 3 minutes; repeat 94° C./70° C. cycling for totalof 5 times C. 94° C. for 2 seconds, 65° C. for 3 minutes; repeat 94°C./65° C. cycling for total of 24 times D. 65° C. for 4 minutes as afinal extension E. 10° C. for an extended incubation    For RT-PCR thecorn inbred line H99 is used and the generic PCR reaction conditions areoutlined below: 1 uL cDNA 5 uL 10x BMB PCR reaction buffer (suppliedwith taq DNA polymerase) 1 uL 10 mM dNTPs 1 uL 10 uM primer 1 1 uL 10 uMprimer 2 0.5 uL taq DNA polymerase (BMB, Indianapolis, IN) 40.5 uL H2O 1uL DMSO (optional)

[0144] 3a. 700353038 Clone ID Analysis and Promoter Isolation

[0145] To determine the distribution of the clone ID 700353038transcripts in corn, RT-PCR is performed using the SEQ ID NO:4 and SEQID NO:5 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, orsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 94° C. 1 minute and 35 cycles of 94° C. 5 seconds, 52° C. 30seconds and 72° C. 30 seconds. PCR products are amplified for this cloneusing cDNA derived from anther, glume/lemma/palea, microspores, andpollen but not with cDNA derived from ear, husk, kernel, meristem,rachis, leaf, root, or silk.

[0146] For the isolation of the clone ID 700353038 promoter, SEQ IDNO:14 is used in combination with SEQ ID NO:1 in a standardGenomeWalker™ PCR reaction with the following conditions: Expand HiFidelity DNA Polymerase (BMB Indianapolis, Ind.) is used in conjunctionwith the supplied buffer #2, 2 μL of a 1:2 dilution of GenomeWalker™libraries made according to the manufacturer's protocol (Clontech, PaloAlto, Calif.) and made with maize genomic DNA. The following cyclingparameters are used: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70°C. 3 min, and 33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For thenested secondary PCR reaction, 1 μL of the primary reaction was usedwith SEQ ID No: 15 and SEQ ID NO: 3 (AP3) in a standard GenomeWalker™PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB Indianapolis,Ind.) with the supplied buffer #2. The reactions are carried out underthe following cycling conditions: 94° C. 1 minute, 5 cycles of 94° C. 4seconds, 72° C. 3 minutes, and 20 cycles of 94° C. 4 seconds and 67° C.3 minutes.

[0147] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis, and bands of approximately 900 bp and800 bp are cut out, purified using the Qiaquick gel extraction kit(Qiagen, Valencia, Calif.) and eluted with 30 μL 10 mM Tris pH. 8.5.Five microliters of the purified band is ligated to 50 ng of pGEM-T-Easyvector (Promega, Madison, Wis.). DNA from individual clones are isolatedusing the Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.) andsequenced using the M13 forward primers and M13 reverse primers.Sequence analysis indicated the two fragments shared 3′ homology butdiverged at their 5′ end. The promoter sequences are shown in SEQ IDNO:80 and SEQ ID NO: 81.

[0148] To determine if the large open reading frames identified in theisolated genomic fragments are transcribed, RT-PCR is performed usingthe following primer pairs: 1. SEQ ID NOS:6 and 7; 2. SEQ ID NOS:8 and9; 3. SEQ ID NOS:10 and 11; and 4. SEQ ID NOS:12 and 13. RT-PCR isperformed using a standard RT-PCR protocol using cDNA derived fromanther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears, leaf,meristem, mature anther/pollen, microspores, rachis, root, or silk. TaqDNA polymerase from BMB (Indianapolis, Ind.) is used in combination withthe supplied reaction buffer. Cycling parameters are as follows: 94° C.1minute and 35 cycles of 94° C. 5 seconds, 52° C. 30 seconds and 72° C.30 seconds.

[0149] Two “ATG” sequences are identified, in frame with an ORF in theEST sequence. Primers are designed to the sequences 5′ to each of theseputative start codons (SEQ ID NO:16 and SEQ ID NO:17). To isolate thepromoter fragment and 5′ leader sequence by PCR, the SEQ ID NO:16 primerand the SEQ ID NO:17 primer are separately used with the AP3 primer (SEQID NO:3) in a standard PCR reaction containing 1 μL of DNA from eitherthe 800 bp or 900 bp clone ID 700353038 promoter fragment, using 0.5 uLBMB taq polymerase and the supplied buffer. The cycling parameters areas follows: 94° C. 1 minute, 20 cycles of 94° C. 5 seconds 55° C. 15seconds and 72° C. 30 seconds.

[0150] The amplified promoter fragments are analyzed by agarose gelelectrophoresis, and bands of approximately 850 bp and 750 bp are cutout and purified using the Qiaquick gel extraction kit (Qiagen,Valencia, Calif.) following the conditions recommended by themanufacturer. The resulting bands are designated SEQ ID NOS: 95 and 96(amplified from SEQ ID NO: 81), and SEQ ID NOS: 97 and 98 (amplifiedfrom SEQ ID NO: 80). The fragments are eluted with 30 μL 10 mM Tris pH.8.5. Five microliter of the purified band is ligated to 50 ng ofpGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individual clonesis isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,Calif.). The DNA is digested with Hind III and Bgl II, and the resultingpromoter fragments are analyzed by agarose gel electrophoresis and bandsof approximately 850 bp and 750 bp are cut out, purified using theQiaquick gel extraction kit (Qiagen, Valencia, Calif.) and eluted with30 μL 10 mM Tris pH. 8.5. Five microliter of the purified band isligated to 1 μL of an expression vector such as pMON19469 shown in FIG.1, that is prepared by digesting 10 μg with BglII and HindIII,separating by agarose gel electrophoresis and purified using theQiaquick gel extraction kit (Qiagen, Valencia, Calif.) and eluted with30 μL 10 mM Tris pH. 8.5. The resulting plasmids contain the promoterfragments, hsp70 intron, and GUS gene and are used to assay promoteractivity of the promoter fragments.

[0151] 3b. 700352826 Clone ID Analysis and Promoter Isolation

[0152] To determine the distribution of the clone ID 700352826transcripts in corn, RT-PCR was performed using the SEQ ID NO:18 and SEQID NO:19 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 94° C. 1 minute and 35 cycles of 94° C. 5 seconds, 52° C. 30seconds and 72° C. 30 seconds. PCR products are obtained with cDNAderived from anther, glume/lemma/palea, micropsores, and pollen but notwith cDNA derived from ear, husk, kernel, meristem, rachis, leaf, root,or silk.

[0153] For the isolation of the clone ID 700352826 promoter, SEQ IDNO:20 is used in combination with SEQ ID NO:1 AP1 in a standardGenomeWalker™ PCR reaction with the following conditions: Expand HiFidelity DNA Polymerase (BMB Indianapolis, Ind.) is used in conjunctionwith the supplied buffer #2, 2 μL of a 1:2 dilution of GenomeWalker™libraries made according to the manufacturer's protocol (Clontech, PaloAlto, Calif.) and made with maize genomic DNA. The following cyclingparameters are used: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70°C. 3 min, and 33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For thenested secondary PCR reaction, 1 μL of the primary reaction is used withSEQ ID NO:21 and SEQ ID NO:3 (AP3) in a standard GenomeWalker™ PCRreaction using Expand Hi Fidelity DNA Polymerase (BMB Indianapolis,Ind.) with the supplied buffer #2. The reactions are carried out underthe following cycling conditions: 94° C. 1 minute, 5 cycles of 94° C. 4seconds, 72° C. 3 minutes, and 20 cycles of 94° C. 4 seconds and 67° C.3 minutes.

[0154] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis and bands of approximately 500 bp and3000 bp are cut out, purified using the Qiaquick gel extraction kit(Qiagen, Valencia, Calif.) and eluted with 30 μL 10 mM Tris pH. 8.5.Five microliters of the purified bands is ligated to 50 ng ofpGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individual clonesis isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.)and is sequenced using M13 forward primers and M13 reverse primers.

[0155] The sequence for clone ID 700352826 is derived from a truncatedcDNA. A BLAST comparison of the promoter sequence against the GenBankdatabase identified polygalacturase clones that allowed thedetermination of the translational initiation codon. SEQ ID NO:22 isdesigned just upstream of the predicted ATG. To isolate the functionalpromoter fragment and 5′ leader sequence by PCR of clone ID 700352826,the SEQ ID NO:22 primer and SEQ ID NO: 3 (AP3)primer are used in astandard PCR reaction containing 1 μL of cloned DNA containing clone ID700352826 promoter fragment, using BMB taq polymerase and the suppliedbuffer. The cycling parameters are as follows: 94° C. 1 minute, 20cycles of 94° C. 5 seconds 55° C. 15 seconds and 72° C. 30 seconds. Thesequence of the promoter is SEQ ID NO:79.

[0156] The amplified promoter fragments are analyzed by agarose gelelectrophoresis and purified using the Qiaquick gel extraction kit(Qiagen, Valencia, Calif.) and eluted with 30 μL 10 mM Tris pH. 8.5 andsubsequently cloned into a plasmid as shown in FIG. 1 (pMON19469).

[0157] 3c. 700354918 Clone Analysis and Promoter Isolation

[0158] To determine the distribution of the clone ID 700354918transcripts in corn, RT-PCR is performed using the SEQ ID NO:23 and SEQID NO:24 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 94° C. 1 minute and 35 cycles of 94° C. 5 seconds, 52° C. 30seconds and 72° C. 30 seconds. PCR products are produced with cDNAderived from anther, glume/lemma/palea, micropsores, and pollen but notwith cDNA derived from ear, husk, kernel, meristem, rachis, root, leaf,or silk.

[0159] For the isolation of the clone ID 700354918 promoter, SEQ IDNO:25 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.) using maize genomic DNA. The following cycling parameters areused: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min, and33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested, secondaryPCR reaction, 1 μL of the primary reaction is used with SEQ ID NO:26 andSEQ ID NO:3 (AP3) in a standard GenomeWalker™ PCR reaction using ExpandHi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the suppliedbuffer #2 . The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes.

[0160] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis and a 700 bp band is isolated, purifiedusing the Qiaquick gel extraction kit (Qiagen, Valencia, Calif.), andeluted with 30 μL 10 mM Tris pH. 8.5. Five microliters of the purifiedband is ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).DNA from individual clones is isolated using the Qiagen Plasmid Mini kit(Qiagen, Valencia, Calif.) and sequenced using the M13 forward primersand M13 reverse primers.

[0161] The clone ID 700354918 sequence is derived from a truncated cDNAand the translation initiation codon was unknown. A BLAST of the 5′ endof the promoter against the GenBank database produced polygalacturaseclones that allowed the prediction of the translational initiation codon67 nucleotides from the 5′ end of promoter. To isolate the functionalpromoter fragment and 5′ leader sequence of clone ID 700354918 by PCR,SEQ ID NO:27 is used in combination with SEQ ID NO: 1 (AP1) in astandard GenomeWalker™ PCR reaction using Expand Hi Fidelity DNAPolymerase (BMB, Indianapolis, Ind.) in conjunction with the suppliedbuffer #2. Each reaction contains 2 μL of a 1:2 dilution ofGenomeWalker™ libraries made according to the manufacturer's protocol(Clontech, Palo Alto, Calif.) and made with maize genomic DNA. Thefollowing cycling parameters are used: 94° C. 1 minute, 7 cycles of 94°C. 4 seconds, 70° C. 3 min, and 33 cycles 94° C. 4 seconds, 68° C. 3minutes. For the nested secondary PCR reaction, 1 μL of the primaryreaction is used with SEQ ID NO:28 and SEQ ID NO:3 (AP3) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) with the supplied buffer #2. The reactions arecarried out under the following cycling conditions: 94° C. 1 minute, 5cycles of 94° C. 4 seconds, 72° C. 3 minutes, and 20 cycles of 94° C. 4seconds and 67° C. 3 minutes.

[0162] The PCR products are purified by gel electrophoresis as describedand cloned into a vector such as shown in FIG. 1 (pMON19469). Thepromoter sequence is shown in SEQ ID NO:82.

[0163] 3d. 700353844 Clone Analysis and Promoter Isolation

[0164] To determine the distribution of the clone ID 700353844transcripts in corn, RT-PCR is performed using the SEQ ID NO:29 and SEQID NO:30 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 94° C. 1 minute and 35 cycles of 94° C. 5 seconds, 52° C. 30seconds and 72° C. 30 seconds. PCR products are obtained with cDNAderived from anther, glume/lemma/palea, micropsores, and pollen but notwith cDNA derived from ear, husk, kernel, meristem, rachis, leaf, root,or silk.

[0165] For the isolation of the clone ID 700353844 promoter, SEQ IDNO:31 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the Genome Walker librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.) using maize genomic DNA. The following cycling parameters areused: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min, and33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested, secondaryPCR reaction, 1 μL of the primary reaction is used with SEQ ID NO:32 andSEQ ID NO:3 (AP3) in a standard GenomeWalker™ PCR reaction using ExpandHi Fidelity DNA Polymerase (BMB Indianapolis, Ind.) with the suppliedbuffer #2 . The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes.

[0166] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis and a 500 bp band is isolated, purifiedusing the Qiaquick gel extraction kit (Qiagen, Valencia, Calif.) andeluted with 30 μL 10 mM Tris pH. 8.5. Five microliters of the purifiedband is ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).DNA from 2 individual clones is isolated using the Qiagen Plasmid Minikit (Qiagen, Valencia, Calif.) and sequenced using the M13 forwardprimers and M13 reverse primers. The sequence of the promoter fragmentis shown in SEQ ID NO:83. The promoter fragment is subsequently clonedinto a plasmid as shown in FIG. 1 (pMON19469).

[0167] 3e. 700355306b Clone Analysis and Promoter Isolation

[0168] To determine the distribution of the clone ID 700355306btranscripts in corn, RT-PCR is performed using the SEQ ID NO:33 and SEQID NO:34 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. 1 minute and 35 cycles of 95° C. 15 seconds, 50° C. 30seconds and 72° C. 30 seconds followed by a 10-minute incubation at 72C. Bands are amplified with cDNA derived from anther, micropsores, andpollen but not with cDNA derived from ear, husk, glume/lemma/palea,kernel, meristem, rachis, leaf, root, or silk.

[0169] For the isolation of the clone ID 700355306b promoter, SEQ IDNO:35 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMBIndianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.) using maize genomic DNA. The following cycling parameters areused: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min, and33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested, secondaryPCR reaction, 1 μL of the primary reaction is used with SEQ ID NO: 36and SEQ ID NO:2 (AP2) in a standard GenomeWalker™ PCR reaction usingExpand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with thesupplied buffer #2 . The reactions are carried out under the followingcycling conditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72°C. 3 minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutesfollowed by 7 minutes at 67° C.

[0170] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis bands are isolated, purified using theQiaquick gel extraction kit (Qiagen, Valencia, Calif.), and eluted with30 μL 10 mM Tris pH. 8.5. To add a HindIII restriction site to the 5′end of the 700355306b promoter fragment, 1 μL of the isolated DNA isamplified under standard GenomeWalker™ PCR conditions using Expand HiFidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the suppliedbuffer #2 in combination with primers SEQ ID NO:37 and SEQ ID NO:3(AP3). The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes followedby 7 minutes at 67° C.

[0171] Twenty five microliters of the tertiary PCR reaction is analyzedby agarose gel electrophoresis, the promoter band is isolated, purifiedusing the Qiaquick gel extraction kit (Qiagen, Valencia, Calif.) andeluted with 30 μL 10 mM Tris pH. 8.5. Five microliters of the purifiedband is ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).DNA from individual clones is isolated using the Qiagen Plasmid Mini kit(Qiagen, Valencia, Calif.) and sequenced by the using the M13 forwardprimers and M13 reverse primers. The sequence of the promoter fragmentis SEQ ID NO:84. The promoter fragment is subsequently cloned into aplasmid as shown in FIG. 1 (pMON19469).

[0172] 3f. 700353142 Clone Analysis and Promoter Isolation

[0173] To determine the distribution of the clone ID 700353142transcripts in corn, RT-PCR is performed using the SEQ ID NO:38 and SEQID NO:39 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. 1 minute and 35 cycles of 95° C. 15 seconds, 50° C. 30seconds and 72° C. 30 seconds followed by a 10-minute incubation at 72°C. Bands are amplified with cDNA derived from anther, micropsores,glume/lemma/palea, husk, meristem, and pollen but not with cDNA derivedfrom ear, kernel, rachis, leaf, root, or silk.

[0174] For the isolation of the clone ID 700353142 promoter, SEQ IDNO:40 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contained 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.) using maize genomic DNA. The following cycling parameters areused: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min, and33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested, secondaryPCR reaction, 1 μL of the primary reaction is used with SEQ ID NO:41 andSEQ ID NO:2 (AP2) in a standard GenomeWalker™ PCR reaction using ExpandHi Fidelity DNA Polymerase (BMB Indianapolis, Ind.) with the suppliedbuffer #2 . The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes followedby 7 minutes at 67° C.

[0175] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis, and PCR products are isolated, purifiedusing the Qiaquick gel extraction kit (Qiagen, Valencia, Calif. cat #28704) and eluted with 30 μL 10 mM Tris pH. 8.5. To add a HindIIIrestriction site to the 5′ end of the 700353142 promoter fragment, 1 μLof the isolated DNA is amplified under standard GenomeWalker™ PCRconditions using Expand Hi Fidelity DNA Polymerase (BMB, Indianapolis,Ind.) with the supplied buffer #2 in combination with primers SEQ IDNO:42 and SEQ ID NO:3 (AP3). The reactions are carried out under thefollowing cycling conditions: 94° C. 1 minute, 5 cycles of 94° C. 4seconds, 72° C. 3 minutes, and 20 cycles of 94° C. 4 seconds and 67° C.3 minutes followed by 7 minutes at 67° C.

[0176] Twenty five microliters of the tertiary PCR reaction is analyzedby agarose gel electrophoresis. The promoter band is isolated, purifiedusing the Qiaquick gel extraction kit (Qiagen, Valencia, Calif.), andeluted with 30 μL 10 mM Tris pH. 8.5. Five microliters of the purifiedband is ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).DNA from individual clones is isolated using the Qiagen Plasmid Mini kit(Qiagen, Valencia, Calif.) and sequenced using the M13 forward primersand M13 reverse primers. The sequence of the promoter is SEQ ID NO:85

[0177] 3g. 700282503 Clone Analysis and Promoter Isolation

[0178] To determine the distribution of the clone ID 700282503transcripts in corn, 2 RT-PCR experiments are performed. In oneexperiment SEQ ID NO: 43 and SEQ ID NO:44 primers are used following astandard RT-PCR protocol using cDNA derived from anther, 6 cm ear,glume/lemma/palea, husk, kernel from 4 cm ears, leaf, meristem, matureanther/pollen, microspores, rachis, root, and silk. Taq DNA polymerasefrom BMB (Indianapolis, Ind.) is used in combination with the suppliedreaction buffer. Cycling parameters are as follows: 95° C. for 1 minuteand 35 cycles of 95° C. 15 seconds, 50° C. 30 seconds and 72° C. 30seconds followed by a 10-minute incubation at 72° C. Bands are amplifiedwith cDNA derived from anther, micropsores, glume/lemma/palea, husk,kernal, and pollen but not with cDNA derived from ear, rachis, leaf,root, meristem, or silk. In the second RT-PCR reaction SEQ ID NO:43 andSEQ ID NO:44 primers are used following a standard RT-PCR protocol usingcDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel from4 cm ears, leaf, meristem, mature anther/pollen, microspores, rachis,root, and silk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is usedin combination with the supplied reaction buffer. Cycling parameters,are as follows: 95° C. 1 minute and 35 cycles of 95° C. 15 seconds, 50°C. 30 seconds and 72° C. 30 seconds followed by a 10-minute incubationat 72° C. Bands are amplified with cDNA derived from anther, kernal, andpollen but not with cDNA derived from ear, rachis, leaf, root,glume/lemma/palea, microspore, husk, meristem, or silk.

[0179] For the isolation of the clone ID 700282503 promoter, SEQ IDNO:46 is used in combination with SEQ ID NO: 1 (AP 1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μl of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.) using maize genomic DNA. The following cycling parameters areused: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min, and33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested, secondaryPCR reaction, 1 μL of the primary reaction was used with SEQ ID NO:47and SEQ ID NO:2 (AP2) in a standard GenomeWalker™ PCR reaction usingExpand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with thesupplied buffer #2. The reactions are carried out under the followingcycling conditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72°C. 3 minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutesfollowed by 7 minutes at 67° C.

[0180] Twenty five microliters of the secondary PCR reaction wasanalyzed by agarose gel electrophoresis and PCR products are isolated,purified using the Qiaquick gel extraction kit (Qiagen, Valencia,Calif.)and eluted with 30 μL 10 mM Tris pH. 8.5. To add a HindIII restrictionsite to the 5′ end of the 700282503 promoter fragment, 1 μL of theisolated DNA is amplified under standard Genome Walker™PCR conditionsusing Expand Hi Fidelity DNA Polymerase (BMB Indianapolis, Ind.) withthe supplied buffer #2 in combination with primers SEQ ID NO:48 and SEQID No:3 (AP3). The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes followedby 7 minutes at 67° C.

[0181] Twenty five microliters of the tertiary PCR reaction is analyzedby agarose gel electrophoresis. A band is isolated, purified using theQiaquick gel extraction kit (Qiagen, Valencia, Calif.) and eluted with30 μL 10 mM Tris pH. 8.5. 5 μL of the purified band is ligated to 50 ngof pGEM-T-Easy vector (Promega, Madison, Wis., Cat. #A1360). DNA fromindividual clones was isolated using the Qiagen Plasmid Mini kit(Qiagen, Valencia, Calif. Cat. #12125) and sequenced using the M13forward primers and M13 reverse primers. The sequence of the promoter isSEQ ID NO:86.

[0182] 3h. 700282409 Clone Analysis and Promoter Isolation (Profilin 2)

[0183] To determine the distribution of the clone ID 700282409transcripts in corn, RT-PCR is performed using the SEQ ID NO:49 and SEQID NO:50 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. for 1 minute and 35 cycles of 95° C. 15 seconds, 50° C.30 seconds and 72° C. 30 seconds followed by a 10 minute incubation at72° C. Bands are amplified with cDNA derived from anther,glume/lemma/palea, meristem, microspore, silk, and pollen but not withcDNA derived from ear, husk, kernel, rachis, leaf, or root.

[0184] For the isolation of the clone ID 700282409 promoter, SEQ IDNO:51 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMBIndianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.) using maize genomic DNA. The following cycling parameters areused: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min, and33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested, secondaryPCR reaction, 1 μL of the primary reaction is used with SEQ ID NO:52 andSEQ ID No. 2 (AP2) in a standard GenomeWalker™ PCR reaction using ExpandHi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the suppliedbuffer #2. The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes followedby 7 minutes at 67° C.

[0185] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis. The bands are isolated, purified usingthe Qiaquick gel extraction kit (Qiagen, Valencia, Calif.) and elutedwith 30 μL 10 mM Tris pH. 8.5. To add a HindIII restriction site to the5′ end of the 700282409 promoter fragment, 1 μL of the isolated DNA isamplified under standard Genome Walker™ PCR conditions using Expand HiFidelity DNA Polymerase (BMB Indianapolis, Ind.) with the suppliedbuffer #2 in combination with primers SEQ ID NO:53 and SEQ ID NO:3(AP3). The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 5 cycles of 94° C. 4 seconds, 72° C. 3minutes, and 20 cycles of 94° C. 4 seconds and 67° C. 3 minutes followedby 7 minutes at 67° C.

[0186] Twenty five microliters of the tertiary PCR reaction is analyzedby agarose gel electrophoresis. A band is isolated, purified using theQiaquick gel extraction kit (Qiagen, Valencia, Calif.) and eluted with30 μL 10 mM Tris pH. 8.5. Five microliters of the purified band isligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.). DNAfrom individual clones is isolated using the Qiagen Plasmid Mini kit(Qiagen, Valencia, Calif.) and sequenced using the M13 forward primersand M13 reverse primers. The sequence designated profilin 2 is shown inSEQ ID NO:87.

[0187] 3i. 700282409 Clone Analysis and Promoter Isolation (Profilin 1)

[0188] All RT-PCR data for 700282409—profilin 1 is the same as theinformation for profilin 2 as described in Example 3h.

[0189] For the isolation of the clone ID 700282409 promoter, SEQ IDNO:51 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 1.5 μL of a 1:2 dilution of the GenomeWalker™libraries made according to the manufacturer's protocol (Clontech, PaloAlto, Calif.) using maize genomic DNA. The following cycling parametersare used: 94° C. 1 minute, 7 cycles of 94° C. 4 seconds, 70° C. 3 min,and 33 cycles 94° C. 4 seconds, 68° C. 3 minutes. For the nested,secondary PCR reaction, 1 μL of the primary reaction is used with SEQ IDNO:54 with SEQ ID No:3 (AP3). The reactions are carried out under thefollowing cycling conditions: 94° C. 1 minute, 5 cycles of 94° C. 4seconds, 72° C. 3 minutes, and 20 cycles of 94° C. 4 seconds and 67° C.3 minutes followed by 7 minutes at 67° C.

[0190] Twenty five microliters of the tertiary PCR reaction is analyzedby agarose gel electrophoresis. A is isolated, purified using theQiaquick gel extraction kit (Qiagen, Valencia, Calif.) and eluted with30 μL 10 mM Tris pH. 8.5. Five microliters of the purified band isligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.). DNAfrom individual clones is isolated using the Qiagen Plasmid Mini kit(Qiagen, Valencia, Calif.) and sequenced using the M13 forward primersand M13 reverse primers. The fall length promoter sequence is shown inSEQ ID NO:88.

[0191] 3j. 700352616 Clone Analysis and Promoter Isolation

[0192] To determine the distribution of the clone ID 700352616transcripts in corn, RT-PCR is performed using the SEQ ID NO:55 and SEQID NO:56 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. 1 minute and 35 cycles of 95° C. 15 seconds, 50° C. 30seconds and 72° C. 30 seconds followed by 10 minutes at 72° C. Bands areamplified with cDNA derived from anther, glume/lemma/palea, andmicrospore but not with cDNA derived from ear, husk, kernel, rachis,leaf, pollen, silk, or root.

[0193] For the isolation of the clone ID 700352616 promoter, SEQ IDNO:58 is used in combination with SEQ ID NO:1 (AP1) in a standard GenomeWalker PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.). The following cycling parameters are used: 94° C. 1 minute, 7cycles of 94° C. 2 seconds, 72° C. 3 min, and 36 cycles 94° C. 2seconds, 66° C. 3 minutes followed by a 4-minute incubation at 66° C.For the nested, secondary PCR reaction, 1 μL of a 1:50 dilution of theprimary reaction is used with SEQ ID NO:59 and SEQ ID NO:2 (AP2) in astandard GenomeWalker™ PCR reaction using Expand Hi Fidelity DNAPolymerase (BMB Indianapolis, Ind.) with the supplied buffer #2. Thereactions are carried out under the following cycling conditions: 94° C.1 minute, 5 cycles of 94° C. 2 seconds, 72° C. 3 minutes, and 25 cyclesof 94° C. 2 seconds and 67° C. 3 minutes followed by 4 minutes at 67° C.

[0194] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis. The bands are isolated, purified usingthe Qiaquick gel extraction kit (Qiagen, Valencia, Calif.), and elutedwith 30 μL ddH2O. Five microliters of the purified band is ligated to 50ng of pGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individualclones is isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,Calif.) and sequenced using the M13 forward primers and M13 reverseprimers.

[0195] To add a HindIII restriction site to the 5′ end and a BamH1 siteat the 3′ end of the 700352616 promoter fragment, 1 μL of the isolatedDNA is amplified under standard Genome Walker™ PCR conditions usingExpand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with thesupplied buffer #2 in combination with primers SEQ ID NO:57 and SEQ IDNO.3 (AP3). The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 3 cycles of 94° C. 2 seconds,70° C. 3minutes and 12 cycles 94° C. 2 seconds 67° C. 3 minutes followed by a4-minute incubation at 67° C. Twenty five microliters of this PCRreaction is analyzed by agarose gel electrophoresis. The bands areisolated, purified using the Qiaquick gel extraction kit (Qiagen,Valencia, Calif.), and eluted with 30 μL ddH2O. Five microliters of thepurified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,Madison, Wis.). DNA from individual clones is isolated using the QiagenPlasmid Mini kit (Qiagen, Valencia, Calif.). The promoter sequence isshown in SEQ ID NO:89.

[0196] The promoter is cloned into a plasmid vector such as shown inFIG. 1 (pMON19469).

[0197] 3k. 700354681 Clone Analysis and Promoter Isolation

[0198] To determine the distribution of the clone ID 700354681transcripts in corn, RT-PCR is performed using the SEQ ID NO:60 and SEQID NO:61 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. 1 minute and 35 cycles of 95° C. 15 seconds, 50° C. 30seconds and 72° C. 30 seconds followed by 10 minutes at 72° C. Bands areamplified with cDNA derived from anther, pollen, and microspore but notwith cDNA derived from ear, glume/lemma/palea, husk, kernel, rachis,leaf, pollen, silk, or root.

[0199] For the isolation of the clone ID 700354681 promoter, SEQ IDNO:62 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.). The following cycling parameters are used: 94° C. 1 minute, 7cycles of 94° C. 2 seconds, 72° C. 3 min, and 36 cycles 94° C. 2seconds, 66° C. 3 minutes followed by a 4-minute incubation at 66° C.For the nested, secondary PCR reaction, 1 μL of a 1:50 dilution of theprimary reaction is used with SEQ ID NO:63 and SEQ ID NO:2 (AP2) in astandard GenomeWalker™ PCR reaction using Expand Hi Fidelity DNAPolymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2. Thereactions are carried out under the following cycling conditions: 94° C.1 minute, 5 cycles of 94° C. 2 seconds, 72° C. 3 minutes, and 25 cyclesof 94° C. 2 seconds and 67° C 3 minutes followed by 4 minutes at 67° C.

[0200] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis. The bands are isolated, purified usingthe Qiaquick gel extraction kit (Qiagen, Valencia, Calif.) and elutedwith 30 μL ddH2O. Five microliters of the purified band is ligated to 50ng of pGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individualclones is isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,Calif.) and sequenced using the M13 forward primers and M13 reverseprimers.

[0201] To add a HindIII restriction site to the 5′ end and Bgl II, BamH1sites at the 3′ end of the 700354681 promoter fragment, 1 μL of theclone harboring the promoter sequence is amplified under standardGenomeWalker™ PCR conditions using Expand Hi Fidelity DNA Polymerase(BMB, Indianapolis, Ind.) with the supplied buffer #2 in combinationwith primers SEQ ID NO:64 and SEQ ID NO:3 (AP3). The reactions arecarried out under the following cycling conditions: 94° C. 1 minute, 3cycles of 94° C. 2 seconds, 70° C. 3 minutes and 12 cycles of 94° C. 2seconds 67° C. 3 minutes followed by a 4-minute incubation at 67° C. A25 μL aliquot of this PCR reaction is analyzed by agarose gelelectrophoresis. The bands are isolated, purified using the Qiaquick gelextraction kit (Qiagen, Valencia, Calif.), and eluted with 30 μL ddH2O.Five microliters of the purified band is ligated to 50 ng of pGEM-T-Easyvector (Promega, Madison, Wis.). DNA from individual clones is isolatedusing the Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.). SEQ IDNO:90 is the promoter sequence for clone ID 700354681 Sequence analysisshows SEQ ID NO:90 to be a fragment of SEQ ID NO: 87.

[0202] The promoter band is purified using the Qiaquick gel extractionkit (Qiagen, Valencia, Calif.), and eluted with 30 μL ddH₂O. An aliquotof the purified band is ligated to an expression vector as shown in FIG.1 (pMON19469) that is prepared for example by digesting 10 μg with BglIIand HindIII, separating by agarose gel electrophoresis and isolating thevector band using the Qiaquick gel extraction kit (Qiagen, Valencia,Calif.), and eluted with 30 μL 10 mM Tris pH 8.5.

[0203] 31. 700353007 Clone Analysis and Promoter Isolation

[0204] To determine the distribution of the clone ID 700353007transcripts in corn, RT-PCR is performed using the SEQ ID NO:65 and SEQID NO:66 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. for 1 minute and 35 cycles of 95° C. 15 seconds, 50° C.30 seconds and 72° C. 30 seconds followed by 10 minutes at 72° C. Bandsare amplified with cDNA derived from anther, glume/lemma/palea, pollen,and microspore but not with cDNA derived from ear, husk, kernel, rachis,leaf, silk, or root.

[0205] For the isolation of the clone ID 700353007 promoter, SEQ IDNO:67 is used in combination with SEQ ID NO:1 AP1 in a standard GenomeWalker PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.). The following cycling parameters are used: 94° C. 1 minute, 7cycles of 94° C. 2 seconds, 72° C. 3 min, and 36 cycles 94° C. 2seconds, 66° C. 3 minutes followed by 4 minute incubation at 66° C. Forthe nested, secondary PCR reaction, 1 μL of a 1:50 dilution of theprimary reaction was used with SEQ ID NO:68 and SEQ ID NO:2 (AP2) in astandard GenomeWalker™PCR reaction using Expand Hi Fidelity DNAPolymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2. Thereactions are carried out under the following cycling conditions: 94° C.for 1 minute, 5 cycles of 94° C. 2 seconds, 72° C. 3 minutes, and 25cycles of 94° C. 2 seconds and 67° C. 3 minutes followed by 4 minutes at67° C.

[0206] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis. The bands are isolated, purified usingthe Qiaquick gel extraction kit (Qiagen, Valencia, Calif.) and elutedwith 30 μl ddH2O. Five microliters of the purified band is ligated to 50ng of pGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individualclones is isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,Calif.), and sequenced using the M13 forward primers and M13 reverseprimers.

[0207] To add a HindIII restriction site to the 5′ end and BglII, BamH1sites at the 3′ end of the 700353007 promoter fragment, 1 μL of theisolated DNA is amplified under standard Genome Walker™ PCR conditionsusing Expand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) withthe supplied buffer #2 in combination with primers SEQ ID NO:69 and SEQID NO:3 (AP3). The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 3 cycles of 94° C. 2 seconds,70° C. 3minutes and 12 cycles 94° C. 2 seconds 67° C. 3 minutes followed by4-minute incubation at 67° C. A 25 μL aliquot of this PCR reaction isanalyzed by agarose gel electrophoresis. The bands are isolated,purified using the Qiaquick gel extraction kit (Qiagen, Valencia,Calif.), eluted and ligated to pGEM-T-Easy vector (Promega, Madison,Wis.). DNA from individual clones is isolated using the Qiagen PlasmidMini kit (Qiagen, Valencia, Calif.). The sequence of the promoterfragment is SEQ ID NO:91.

[0208] The promoter fragment is cloned into a vector such as shown inFIG. 1 (pMON19469) containing the hsp70 intron/GUS cassette.

[0209] 3m. 700352625 Clone Analysis and Promoter Isolation

[0210] To determine the distribution of the clone ID 700352625transcripts in corn, RT-PCR is performed using the SEQ ID NO:70 and SEQID NO:71 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. for 1 minute and 35 cycles of 95° C. 15 seconds, 50° C.30 seconds and 72° C. 30 seconds followed by 10 minutes at 72° C. Bandsare amplified with cDNA derived from anther, glume/lemma/palea, pollen,and microspore but not with cDNA derived from ear, husk, kernel, rachis,leaf, silk, or root.

[0211] For the isolation of the clone ID 700352625 promoter, SEQ IDNO:72 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.). The following cycling parameters are used: 94° C. 1 minute, 7cycles of 94° C. 2 seconds, 72° C. 3 min, and 36 cycles 94° C. 2seconds, 66° C. 3 minutes followed by 4 minute incubation at 66° C. Forthe nested, secondary PCR reaction, 1 μL of a 1:50 dilution of theprimary reaction was used with SEQ ID NO:73 and SEQ ID NO. 2 (AP2) in astandard GenomeWalker™ PCR reaction using Expand Hi Fidelity DNAPolymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2. Thereactions are carried out under the following cycling conditions: 94° C.1 minute, 5 cycles of 94° C. 2 seconds, 72° C. 3 minutes, and 25 cyclesof 94° C. 2 seconds and 67° C. 3 minutes followed by 4 minutes at 67° C.

[0212] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis. The bands are isolated, purified usingthe Qiaquick gel extraction kit (Qiagen, Valencia, Calif.) and elutedwith 30 μL ddH2O. Five microliters of the purified band is ligated to 50ng of pGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individualclones is isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,Calif.), and sequenced using the M13 forward primers and M13 reverseprimers. To add a HindIII restriction site to the 5′ end and a BamH1site at the 3′ end of the 700352625 promoter fragment, 1 μL of theisolated DNA was amplified under standard Genome Walker™ PCR conditionsusing Expand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) withthe supplied buffer #2 in combination with primers SEQ ID NO:74 and SEQID NO:3 (AP3). The reactions are carried out under the following cyclingconditions: 94° C. 1 minute, 3 cycles of 94° C. 2 seconds,70° C. 3minutes and 12 cycles 94° C. 2 seconds 67° C. 3 minutes followed by4-minute incubation at 67° C. Twenty five microliters of this PCRreaction is analyzed by agarose gel electrophoresis. The bands areisolated, purified and ligated to pGEM-T-Easy vector DNA (Promega,Madison, Wis.). DNA from individual clones is isolated using the QiagenPlasmid Mini kit (Qiagen, Valencia, Calif.). The promoter sequence isshown in SEQ ID NO:92. The promoter fragment is cloned in an expressionvector as shown in FIG. 1 (pMON19469).

[0213] 3n. 700382630 Clone Analysis and Promoter Isolation

[0214] To determine the distribution of the clone ID 700382630transcripts in corn, RT-PCR is performed using the SEQ ID NO:75 and SEQID NO:76 primers following a standard RT-PCR protocol using cDNA derivedfrom anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,leaf, meristem, mature anther/pollen, microspores, rachis, root, andsilk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used incombination with the supplied reaction buffer. Cycling parameters are asfollows: 95° C. for 1 minute and 35 cycles of 95° C. 15 seconds, 50° C.30 seconds and 72° C. 30 seconds followed by 10 minutes at 72° C. Bandsare amplified with cDNA derived from anther, glume/lemma/palea, pollen,and microspore but not with cDNA derived from ear, husk, kernel, rachis,leaf, silk, or root.

[0215] For the isolation of the clone ID 700382630 promoter, SEQ IDNO:77 is used in combination with SEQ ID NO:1 (AP1) in a standardGenomeWalker™ PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,Indianapolis, Ind.) in conjunction with the supplied buffer #2. Eachreaction contains 2 μL of a 1:2 dilution of the GenomeWalker™ librariesmade according to the manufacturer's protocol (Clontech, Palo Alto,Calif.). The following cycling parameters are used: 94° C. 1 minute, 7cycles of 94° C. 2 seconds, 72° C. 3 min, and 36 cycles 94° C. 2seconds, 66° C. 3 minutes followed by 4 minute incubation at 66° C. Forthe nested, secondary PCR reaction, 1 μL of a 1:50 dilution of theprimary reaction is used with SEQ ID NO:78 and SEQ ID NO:3 (AP3) in astandard GenomeWalker™ PCR reaction using Expand Hi Fidelity DNAPolymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2. Thereactions are carried out under the following cycling conditions: 94° C.1 minute, 5 cycles of 94° C. 2 seconds, 72° C. 3 minutes, and 25 cyclesof 94° C. 2 seconds and 67° C. 3 minutes followed by 4 minutes at 67° C.

[0216] Twenty five microliters of the secondary PCR reaction is analyzedby agarose gel electrophoresis. The bands are isolated, purified, andligated to pGEM-T-Easy vector (Promega, Madison, Wis.) DNA. DNA fromindividual clones is isolated using the Qiagen Plasmid Mini kit (Qiagen,Valencia, Calif.), and sequenced using the M13 forward primers and M13reverse primers. The promoter sequence is shown in SEQ ID NO:93.

Example 4

[0217] Promoter Isolation and Cloning

[0218] The DNA fragments resulting from the nested PCR amplificationdescribed above are isolated and gel purified. A 25 μL aliquot of thesecondary PCR is run on an agarose gel. The DNA fragment of thesecondary PCR product is purified from the agarose gel using the QiagenKit following the conditions suggested by the manufacturer. The purifiedDNA is ligated to pGEM-T Easy vector (pGEM-T Easy Vector System I,Promega Corp., Madison, Wis.) following the conditions recommended bythe manufacturer. An aliquot of the ligation reaction is transformedinto a suitable E. coli host such as DH10B and the cells plated onselection medium (for DH10B, 100 g/mL carbenicillin). Bacterialtransformants are selected, grown in liquid culture, and the plasmid DNAisolated using a commercially available kit such as the Qiaprep SpinMicroprep Kit (Qiagen Corp., Valencia, Calif.). Purified plasmidcontaining the predicted insert size based on restriction enzymeanalysis are sequenced using the dye terminator method in bothdirections using the M13 forward and reverse primers shown in SEQ IDNO:4 (M13 forward primer) and SEQ ID NO:5 (M13 reverse primer).Restriction enzymes are available from a number of manufacturers (see,for example, Boehringer Mannheim (Indianapolis, Ind.). The 5′ flankingregions containing the promoter sequences are determined and shown inSEQ ID NOS:79-98. Engineering restriction sites for cloning intosuitable vectors is done using standard molecular biology techniquesknown to those skilled in the art.

Example 5

[0219] Transient Analysis of Promoter Activity in Protoplasts andMicrospores

[0220] For transient expression, promoter fragments are cloned intoexpression vectors such as pMON19469 shown in FIG. 1. Plasmid pMON19469is an expression vector consisting of the following genetic components:P-e35S is the promoter for the 35S RNA from CaMV containing aduplication of the −90 to −300 region; HSP70 intron is the interveningsequence of the maize heat shock protein as described in U.S. Pat. Nos.5,593,874 (herein incorporated by reference in its entirety) and5,859,347 (herein incorporated by reference in its entirety); GUS:1 isthe coding region for beta-glucuronidase; nos 3′ is the terminationsignal from the nopaline synthase gene; ori-M13 and ori-pUC are originsof replication; AMP is the coding region for ampicillin selection. If atranslational start codon of a target promoter is identified, thefragment is cloned into pMON19469 in place of the P-e35S geneticelement. If an AUG is not identified, the promoter fragment is clonedinto an expression vector modified to enable translational fusions witha reporter gene such as β-glucuronidase (GUS) (Jefferson et al., EMBOJ., 6:3901, 1987) or green fluorescent protein (GFP) as described inPang et al. (Plant Physiol. 112:893, 1996).

[0221] The expression constructs are tested in a transient plant assay.A number of assays are available and known to those skilled in the art.Analysis of reporter genes in a protoplast system can be used to assessthe activity of a regulatory element, such as a promoter operably linkedto the reporter gene. A leaf protoplast isolation and electroporationprotocol is followed essentially as described by Sheen (The Plant Cell3:225-245, 1991) with the following modifications: the seed used isFR27RHM×FRMol7RHM from Illinois Foundation Seeds. The seed is surfacesterilized for 2 minutes in 95% ethanol, rinsed twice with sterilewater, 30 minutes in 50% bleach (Clorox) plus 2 drops of Tween-20, threerinses in sterile water followed by a 5-minute soak in benlate/captansolution to prevent fungal growth. The seeds are germinated inphytotrays containing 100 ml ½ MS media (2.2 g/L MS salts, 0.25%gelrite), 7 seeds per phytotray. The seeds are grown 5 days at 26° C. in16/8 hour day/night photoperiod and 7 days in the dark at 28° C. Thesecond leaf from each plant is sliced longitudinally using Feather No.11 surgical blades. Digestion time is two hours and 10 minutes in thelight at 26° C. After digestion, the plates are swirled two times at80-100 rpm for 20 seconds each and the protoplast/enzyme solution ispipetted through a 190 μm tissue collector. Protoplasts are countedusing a hemacytometer counting only protoplasts that are intact andcircular. Ten to fifty micrograms of DNA containing the vector ofinterest is added per cuvette. Final protoplast densities atelectroporation range from 3×10⁶/mL to 4.5×10⁶/mL. Electroporations areperformed in the light using Bio-Rad Gene pulser cuvettes (Bio-RadHercules, Calif.) with a 0.4 cm gap and a maximum volume of 0.8 mL at125 μFarads capacitance and 260 volts. The protoplasts are incubated onice after resuspension in electroporation buffer and are kept on ice incuvettes until 10 minutes after electroporation. The protoplasts arekept at room temperature for ten minutes before adding 7 mL ofprotoplast growth medium. The protoplast culture medium has beendescribed (Fromm et al., Methods in Enzymology 153, 351-366, 1987).Culture plates are layered with the growth medium and 1.5% SeaPlaqueagarose (FMC BioProducts, Rockland, Me.) to prevent protoplast loss.Samples are cultured in the light at 26° C., 16/8 day/night cycle, untilharvested for the assay (typically 18-22 hours after electroporation).Samples are pipetted from the petri plates to 15 mL centrifuge tubes andharvested by centrifugation at 800-1000 rpm. The supernatant is removedand samples are assayed immediately for the gene of interest. Samplescan also be frozen for later analysis.

[0222] For analysis of promoter activity in a wheat protoplast system,the method for isolation and preparation of wheat protoplasts isperformed as described by Zhou et al. (Plant Cell Reports 12:612, 1993).The electroporation buffer used has been described (Li et al., 1995).The culture medium used is MS1 MSM (4.4 g Gibco MS salts/L, 1.25 mlThiamine HCL (0.4 mg/mL), 1 mL 2,4-D (1 mg/mL), 20 g/L sucrose, 0.15 mLasparagine (15 mg/mL), 0.75 g MgCl₂. 109 g/L 0.6M mannitol, pH5.5.Mustang protoplasts are used for protoplast isolation about four daysafter subculture. Briefly, 8 g of wheat cell suspension is poured into aculture tube, the cells are allowed to settle. The medium is removed andremaining cells are resuspended with 40 mL enzyme solution, transferredto a petri plate, wrapped in foil, and incubated at 26° C. for 2 hourson a rotator at 40 rpm. The suspension is centrifuged at 200 g for 8min., washed twice with centrifugation between each wash, resuspended in10 mL wash solution and stored on ice. The number of protoplasts isdetermined and the volume adjusted to a final concentration of 4×10⁶protoplasts/ml. About 0.75 mL of protoplasts is added to eachelectroporation cuvette and up to about 50 μg plasmid DNA of the vectorin 50 μL solution is added to the protoplasts. The electroporationconditions are 960 μFarads and 160 volts using a Bio-Rad Gene Pulser(Bio-Rad Laboratories, Hercules, Calif.). The samples remain on ice for10 minutes prior to and during electroporation. After electroporation,the samples are left on ice for about 10 minutes and removed and allowedto warm to room temperature for 10 minutes. The electroporated cells arepipetted into MS1 WSM medium and incubated in the dark for 18-22 hoursat 24° C. The cells are harvested by centrifugation at 200-250 g for 8minutes and frozen on dry ice for subsequent analysis of expression ofthe gene of interest.

[0223] In another transient assay system, barley microspores are used.For this assay shoots are collected and spikes are removed from thesheath and placed in 15×100 mm plates. Fifteen microliters of 0.3 M icecold mannitol is added into each plate containing 10 spikes. The platesare sealed with parafilm and kept at 4° C. for 3-4 days. Pre-treatedspikes are cut about 1-2 cm into a chilled blender cup (about 10two-rowed spikes needed/plate). The spikes are covered with enough coldmannitol to create a slurry and blended at low speed in a Waring blenderfor 6-10 seconds. The slurry is filtered through cheesecloth or a nylonmembrane and the filtrate is filtered through a 100μ mesh nylon membraneinto a 50 mL centrifuge tube. The mixture is spun for 5 minutes at 900rpm and the liquid is decanted and microspore pellet resuspended in 2 mLliquid FHG medium. The microspores are filtered through three layers ofWhatman #2 filter paper into a filtering flask under vacuum. About 2 mLmicrospores are dropped on each filter set and the uppermost filterpaper is transferred to solid medium (FHG+0.25 M mannitol+0.25 Msorbitol on a 15×100 mm plate). The plates are sealed with parafilm andincubated in the dark at 25° C. for 3-4 hours prior to particlebombardment. A number of methods of particle bombardment can be used(see, for example, Klein et al., Bio/Technology 6:559, 1988; Christou,Particle Bombardment for Genetic Engineering of Plants, Academic Press,1996). After bombardment, the plates are sealed and kept at 25° C. for20-24 hours. 0.3 M mannitol solution is used to wash microspores fromthe filter paper and the microspores are collected by centrifugation andanalyzed for expression of the gene of interest. The FHG medium recipeis as follows: Macroelements (mg/L) include, 1900 mg KNO, 165 mg NH₄NO₃,170 mg KH₂PO₄, 370 mg MgSO₄ 7 H₂O, 440 mg CaCl₂ 2H₂O; Microelements(mg/L) include 40 mg FeNa.EDTA, 22.3 mg MnSO₄.5H₂O, 6.2 mg H₃B0₃, 8.6 mgZnSO₄, 0.025 CuSO₄.5H₂O, 0.25 mg NaM004.2H₂O.

Example 6

[0224] Transient Analysis of Promoter Activity in Wheat ReproductiveTissues

[0225] For analysis of promoter activity in wheat reproductive tissuessuch as wheat anthers and ovaries, constructs containing the potentialpromoter, the HSP70 intron and the GUS gene are bombarded into wheatanthers and ovaries from wheat spikes in which the boot is justbeginning to open. One spike of anthers and ovaries is dissected perplate (1 liter plate medium containing 4.4 g MS salt, 40 g maltose, 40 graffinose, 22.78 g mannitol, 1.95 g MES and 4 g phytagel at pH 5.8). Twoand a half micrograms of each DNA sample (1 μg/μL) to be tested isprecipitated with 12.5 μL tungsten, 5.0 μL 0.1M spermidine and 12.5 μL1.0M calcium chloride for 40 minutes at room temperature. For gunpowderbombardments, 12.5 μL of the supernatant is removed and remainder of thesample is sonicated before each shot. Two and a half microliters of theDNA precipitant is bombarded per shot. For the helium gun bombardments,the precipitated DNA is spun down, washed with 70% EtOH and with 100%EtOH and resuspended in 40 μL 100% EtOH. Five microliters of the DNA isbombarded per plate. For either method, each plate is shot twice, andtwo plates are assayed per DNA sample. After bombardment, the plates areincubated overnight at 24° C. in the dark. The next day the anther andovaries are transferred to a GUS staining solution. To increase thepenetration of the staining solution, the samples are put in a vacuumchamber for 10 minutes. Anthers and ovaries are incubated in thestaining solution at 37° C. for 16-24 hours. The staining solution isreplaced with 70% Ethanol, and the tissues are stored at 4° C. Stainingis strictly qualitative, either there is expression or not. The stainingindicates nothing of the tissue specificity of the potential promotersin stable plates, because the wheat ovary is a very promiscuous tissuethat allows many active promoters to be expressed in this transientsystem.

Example 7

[0226] Promoter Activity from Transient Assay Analysis

[0227] In general, transcriptional regulatory elements necessary forpromoter activity are located within a few hundred bases of thetranscriptional start site. In many plant promoters, regulatory elementssufficient for driving heterologous gene expression in a spatial andtemporal pattern that mimics the expression of the endogenous gene arelocated within 1000 base pairs 5′ of the transcriptional start site.There are some genes, however, where transcriptional regulatory elementscan be located kilobases away, 5′ or 3′ to the transcriptional startsite.

[0228] The transient assay is a system well suited to determining ifsufficient regulatory elements are present for transcription initiation.As described herein, a DNA fragment is operationally linked to areporter gene of interest and that construct is “placed” (throughparticle bombardments, electroporation, etc) into cells, protoplasts ortissues. Expression of the reporter gene in the recipient cellsindicates that enough regulatory sequences reside in the DNA fragment toinitiate transcription. Thereby, the DNA fragment can be considered apromoter. The transient assay does not provide any data regarding thepattern of gene expression the promoter fragment would provide in vivo.A prediction of the promoter's activity can be made based on the patternof the endogenous gene's activity, but the accuracy of this predictionis dependent on whether the promoter fragment contains all the necessaryregulatory elements responsible for the proper expression of theendogenous gene.

[0229] A negative result in a transient assay does not necessarilyindicate that a tested DNA fragment has no promoter activity. Inaddition to experimental error, some of the conditions which couldresult in a negative result are: 1). A translational start codon islocated within the DNA fragment thereby blocking expression of thereporter gene; 2). The DNA fragment contains a transcriptional startsite and a splice donor site, but lacks a splice acceptor site.Therefore, the reporter gene is not expressed because the message is notproperly spliced; 3). Transcription factors specific to the function ofthe promoter region may not be present in the tissues used for thetransient assay; or 4). The level of transcription is below the limitsof detection of the assay.

[0230] To test the DNA fragments contained in Example 3 for promoteractivity, SEQ ID NOS: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97 and 98 are assayed by particle bombardment of barleymicrospores (see Example 5) or particle bombardment of wheatreproductive tissues (see Example 6). As described herein, theexperimental cassette used to test each putative promoter fragmentcontains the fragment operationally linked to the hsp70 intron and theGUS gene. The experiment for each construct is carried out at least 2times. A construct containing the e35S promoter operationally linked tothe hsp70 intron and GUS gene is used as a positive control and a no-DNAbombardment is used as a negative control. The data are summarized inTable 2. Table 2. Summary of transient assay data testing for promoteractivity of the DNA fragments containing SEQ ID NOS: 82-98. In the firstcolumn are the Clone IDs of the EST sequences used to isolate thepromoter fragments (see Example 3). The second column are the SEQ IDnumbers of the fragments tested in the transient assay. The constructnames are listed in the third column. Each construct contains a fragmentoperably linked to the hsp70 intron and GUS gene. The Fourth columnindicates the transient assay used. The fifth column is the level of GUSactivity detected based on a qualitative evaluation of the number andintensity of positively staining cells in the assay. Low indicates muchlower than 35S, medium indicates slightly lower than 35S, and highindicates greater than or equal to 35S. SEQ ID Level Clone ID #Construct Transient Assay Detected 700353038 96 pMON48133 wheatreproductive low tissue 98 pMON48132 wheat reproductive low tissue 95pMON48131 wheat reproductive low tissue 97 pMON48130 wheat reproductivelow tissue 700352826 94 pMON48136 wheat reproductive medium tissue700354918 82 pMON48134 wheat reproductive not tissue detected 70035384483 pMON48137 wheat reproductive medium tissue 700355306 84 pMON53312wheat reproductive not tissue detected 700353142 85 pMON53306 wheatreproductive low tissue 700282503 86 pMON53320 wheat reproductive nottissue detected 700282409 88 pMON53310 wheat reproductive high tissue700282409 87 pMON53308 wheat reproductive medium tissue 700352616 89pMON49324 wheat reproductive low tissue 89 pMON49323 wheat reproductivenot tissue detected 89 pMON49322 wheat reproductive not tissue detected700354681 90 pMON49300 wheat reproductive high tissue 700353007 91pMON49332 wheat reproductive low tissue 91 pMON49327 wheat reproductivenot tissue detected 91 pMON49326 wheat reproductive low tissue 91pMON49325 wheat reproductive low tissue 700352625 92 pMON49337 wheatreproductive low tissue 92 pMON49330 wheat reproductive low tissue 92pMON49329 wheat reproductive not tissue detected 92 pMON49328 wheatreproductive low tissue 700382630 93 pMON49336 barley microspore low

[0231] 7a: 700353038

[0232] For clone ID 700353038, two promoter fragments are isolated thatcomprise SEQ ID NOS:80 and 81. Two potential translational start codons(ATG) are identified at the 3′ end of each fragment. Therefore, two newfragments are generated from each fragment which has 3′ ends justupstream of one of the ATG's (see Example 3A). These new promoterfragments comprise SEQ ID NOS:95 and 96 (derived from SEQ ID NO:81) andSEQ ID NOS:97 and 98 (derived from SEQ ID NO:80). Each DNA fragmentcomprising each of SEQ ID NOS: 95, 96, 97, and 98 operably linked to thehsp70 intron and the GUS gene to generate constructs pMON48131,pMON48133, pMON48130 and pMON48132, respectively. These constructs aretested in the wheat reproductive tissue transient assay. GUS activity isdetected for each construct. The level of expression detected is muchlower than seen with e35S but greater than that seen with the no DNAbombardments. These data indicate that SEQ ID NOS:95, 96, 97, and 98contain promotor activity and translation initiated within the GUS gene.

[0233] 7b: 700352826

[0234] The fragment comprising SEQ ID NO:79 has an internal HindIII siteat position 416. SEQ ID NO: 94 contains the sequences 417-2213 of SEQ IDNO:79 (all sequences 3′ of the HindIII site, inclusive). The fragmentcomprising SEQ ID NO:94 is operably linked to the hsp70 intron and theGUS gene to generate the construct pMON48136. This construct is testedfor promotor activity using the wheat reproductive tissue transientassay. GUS activity is detected at a level slightly below that seen withthe e35S positive control indicating that SEQ ID NO:94 has promotoractivity.

[0235] 7c: 700354918

[0236] The fragment comprising SEQ ID NO:82 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON48134. Thisconstruct is tested for promoter activity using the wheat reproductivetissue transient assay. GUS activity is not detected which could be dueto any of the reasons described above.

[0237] 7d: 700353844

[0238] The fragment comprising SEQ ID NO:83 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON48137. Thisconstruct is tested for promoter activity using the wheat reproductivetissue transient assay. GUS activity is detected at a level slightlybelow that seen with the e35S positive control indicating that SEQ IDNO:83 has promoter activity.

[0239] 7e: 700355306

[0240] The fragment comprising SEQ ID NO:84 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON53312. Thisconstruct is tested for promoter activity using the wheat reproductivetissue transient assay. GUS activity is not detected which could be dueto any of the reasons described above.

[0241] 7f: 700353142

[0242] The fragment comprising SEQ ID NO:85 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON53306. Thisconstruct is tested in the wheat reproductive tissue transient assay.GUS activity is detected at a level of expression much lower than seenwith e35S but greater than that seen with the no DNA bombardmentsindicating that SEQ ID NO:85 has promoter activity.

[0243] 7g: 700282503

[0244] The fragment comprising SEQ ID NO:86 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON53320. Thisconstruct is tested for promoter activity using the wheat reproductivetissue transient assay. GUS activity is not detected which could be dueto any of the reasons described above.

[0245] 7h: 700282409 Profilin 2 and 700354681

[0246] The fragment comprising SEQ ID NO:90 is a smaller version of theputative promoter comprising SEQ ID NO:87. The fragment comprising SEQID NO:90 is operably linked to the hsp70 intron and the GUS gene togenerate the construct pMON449300. The fragment comprising SEQ ID NO:87is operably linked to the hsp70 intron and the GUS gene to generate theconstruct pMON53308. Both constructs are tested in the wheatreproductive tissue transient assay. GUS activity is detected with eachfragment tested. GUS activity is detected at a level slightly below thatseen with the e35S positive control with SEQ ID NO:87. Gus activity isdetected at a level equal to that seen with the e35S positive controlwith SEQ ID NO:90. These data indicate that both SEQ ID NOS:87 and 90contain promoter activity.

[0247] 7i: 700282409 Profilin 1

[0248] The fragment comprising SEQ ID NO:88 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON53310. Thisconstruct is tested in the wheat reproductive tissue transient assay..Gus activity is detected at a level equal to that seen with the e35Spositive control indicating that SEQ ID NO:88 has promoter activity.

[0249] 7j: 700352616

[0250] The translational start codon is not readily identifiable in theEST sequence for clone ID 7003542616. Therefore, the fragment comprisingSEQ ID NO:89 is placed in vectors designed to generated GUS fusions (seeExample 5). Three constructs are generated, pMON49322, pMON49323, andpMON49324 each representing a different reading frame for a putativetranslational fusion between the sequences in SEQ ID NO:89 and GUS. Allthree constructs are tested in the wheat reproductive tissue transientassay. GUS activity is detected with only one construct, at a level ofexpression much lower than seen with e35S but greater than that seenwith the no DNA bombardments. These data indicate that a translationalstart codon is located within SEQ ID NO:89 and that SEQ ID NO:89 haspromoter activity.

[0251] 7k: 700353007

[0252] The translational start codon is not readily identifiable in theEST sequence for clone ID 700353007. Therefore, the fragment comprisingSEQ NO: ID 92 is placed in vectors designed to generate GUS fusions (seeExample 5). Three constructs are generated, pMON49325, pMON49326, andpMON49327, each representing a different reading frame for a putativetranslational fusion between the sequences in SEQ ID NO:91 and GUS. Allthree constructs are tested in the wheat reproductive tissue transientassay. GUS activity is detected with two constructs, at a level ofexpression much lower than seen with e35S but greater than that seenwith the no DNA bombardments. This indicates that translation doesinitiate off the translational start codon located within the GUS gene.To test that, SEQ ID NO:91 is tested using the conventional vectordescribed in Example 5, which has an inframe STOP codon upstream of theGUS translational start codon. With this construct, pMON 49332, GUSactivity is detected at a level of expression much lower than seen withe35S but greater than that seen with the no DNA bombardments. These dataindicate that SEQ ID NO:91 has promoter activity.

[0253] 7l: 700352625

[0254] The translational start codon is not readily identifiable in theEST sequence for clone ID 700352625. Therefore, the fragment comprisingSEQ ID NO:92 is placed in vectors designed to generate GUS fusions (seeExample 5). Three constructs are generated, pMON49328, pMON49329, andpMON49330, each representing a different reading frame for a putativetranslational fusion between the sequences in SEQ ID NO:92 and GUS. Allthree constructs are tested in the wheat reproductive tissue transientassay. GUS activity is detected with two constructs, at a level ofexpression much lower than seen with e35S but greater than that seenwith the no DNA bombardments. This indicates translation is initiatingat the translational start codon within the GUS gene. To test that, SEQID NO:92 is tested using the conventional vector described in Example 5,which has an in frame STOP codon upstream of the GUS translational startcodon. With this construct, pMON49337, GUS activity is detected at alevel of expression much lower than seen with e35S but greater than thatseen with the no DNA bombardments indicating that SEQ ID NO:92 haspromoter activity.

[0255] 7m: 700382630

[0256] The fragment comprising SEQ ID NO:93 is operably linked to thehsp70 intron and the GUS gene to generate the construct pMON49336. Thisis tested in the barley microspore transient assay. GUS activity isdetected at a level of expression much lower than seen with e35S butgreater than that seen with the no DNA bombardments indicating that SEQID NO:93 has promoter activity.

Example 8

[0257] Promoter Activity Analysis in Plants

[0258] For stable plant transformation the 5′ regulatory sequences arecloned into a plant transformation vector such as shown in FIG. 2.Plasmid pMON51850 is a double border (right and left T-DNA borders)plant transformation vector and contains the following geneticcomponents: NOS 3′ is the termination signal from the nopaline synthasegene; ori-322 and ori-V are origins of replication; kan is the codingregion for kanomycin selection.

[0259] The promoter is operably linked to any gene of interest such as aglyphosate tolerance gene along with other regulatory sequencesincluding, but not limited to, non-translated leaders and terminators asdescribed above, and transformed into a target crop of interest via anappropriate delivery system such as Agrobacterium-mediatedtransformation (see, for example, U.S. Pat. No. 5,569,834, hereinincorporated by reference in its entirety, U.S. Pat. No. 5,416,011,herein incorporated by reference in its entirety, U.S. Pat. No.5,631,152, herein incorporated by reference in its entirety, U.S. Pat.No. 5,159,135, herein incorporated by reference in its entirety and U.S.Pat. No. 5,004,863, herein incorporated by reference in its entirety) orparticle bombardment methods (see, for example, Patent Applns. WO92/15675. WO 97/48814 and European Patent Appln. 586,355, and U.S. Pat.Nos. 5,120,657, 5,503,998, 5,830,728 and 5,015,580, all of which areherein incorporated by reference in their entirety). A large number oftransformation and regeneration systems and methods are available andwell-known to those skilled in the art. The stably transformed plantsand progeny are subsequently analyzed for expression of the gene intissues of interest by any number of molecular, immunodiagnostic,biochemical, and/or field evaluation methods known to those skilled inthe art.

[0260] The results from the transient assay analysis described inExamples 7 are qualitative regarding the promoter activity of the DNAfragments tested. Although these promoters are predicted to be active inmale reproductive tissues, the transient assay analysis does notdemonstrate any support for this. To determine the promoter activity inmale reproductive tissues, the promoter fragments are cloned upstream ofa gene of interest (either GUS or the MS2 coat protein), placed in aplant transformation vector and transformed into plants. Tissues from R0plants are harvested and assayed for the gene of interest.

[0261] Detection of GUS activity in male reproductive tissues isdescribed in Example 5. For the detection of the MS2 coat protein,anther extracts are analyzed by immunodetection on Western Blots orELISA analysis. For western blots, the T7 tag monoclonal and horseradish peroxidase conjugated antibody (Novagen, Madison, Wis.) is usedto detect MS2 protein expression in anther tissues. Total protein isextracted (extraction buffer containing 1× PBS and .01% Tween-20) fromanther, 10 μg of total protein sample is separated on a 10-20%polyacrylamide gradient gel (BioRAD, Hercules, Calif.) and transferredonto ECL nitrocellulose membrane (Amersham, Arlington, Ill.). A 1:5000dilution of primary antisera is used to detect ACOX protein using theECL detection system (Amersham, Arlington, Ill.). A 15 Kd protein isdetected.

[0262] For ELISA quantification of MS2 coat protein levels in anthers,crude anther extracts containing 1 ug total protein is added to a96-well Nunc-Immuno MaxiSorb plate coated with 100 μl of purifiedpolyclonal anti-MS2 coat protein IgG antibody (0.1 ng/μL) in coatingbuffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6). The plate is sealed andincubated at 37° C. for one hour. The plate is then rinsed three timeswith washing buffer (1× PBS, 0.05% Tween 20, pH7.4). Fifty microlitersof the anther extract containing 1 μg of total protein is added to awell, followed by addition of 50 μl of a 1:10,000 dilution of anti-T7tag monoclonal and HSP conjugated antibody (Novagen). The loaded plateis incubated at 37° C. for one hour then rinsed three times with washingbuffer. To develop the plate, 100 μl of substrate (a 1:1 mixture of H₂O₂and TMB (3,3′-5,5′-tetra methyl benzidine), Kirgeggard and Perry,#50-76-03, Gaithersburg, Md.) is added to each well and the plate isincubated at room temperature for 3-5 min. One hundred microliters ofstop solution (3M H₃PO₄) is added to terminate the reaction. The plateis read on a Spectra Max 340 (Molecular Devices, Sunnyvail, Calif.) at450 nm.

[0263] Plasmids can be transformed into either monocot or dicot plants.The promoter fragments are derived from corn (Zea mays). Therefore,activity in a dicot plant would indicate a broad spectrum of plants inwhich the promoter is active. The dicot plant Arabidopsis thalianaoffers several advantages as a model system to study promoter systems:ease of transformation, quick life cycle, and multiple stages of floraldevelopment on each plant. Monocot promoters that are active inArabidopsis anthers are likely to be active in many monocot and dicotspecies. To test this, some promoters which are active in Arabidopsisare also tested in monocot plants. As shown below and summarized inTable 3, all promoter fragments tested that are active in Arabidopsisare also active in monocots. Table 3. Summary of promoter activity instably transformed plants. In the first column are the Clone IDs of theEST sequences used to isolate the promoter fragments (see Example 3).The second column shows the SEQ ID numbers of the fragments tested inthe transient assay. The third column lists the introns used in theconstructs. No introns are used in constructs for dicot transformation.The fourth column lists the reporter genes used in the constructs. Thefifth column show the construct names. In the sixth column are theorganisms transformed. The seventh column shows the type of assay usedto detect the reporter gene. The eighth column shows the number ofplants assayed. The ninth column shows the number of plants showing maleexpression and the last column describes any other tissues where thereporter protein is detected. Num

Number of Sho

Plants M

Clone ID SEQ ID Intron Gene Assayed Construct Organism Assay TypeAssayed Expr

700353038 98 none GUS pMON48183 Arabidopsis GUS activity 4

98 hsp70 intron MS2 coat protein pMON42438 rice Western 5

700352826 94 none GUS pMON48185 Arabidopsis GUS activity 4

700353844 83 none GUS 48186 Arabidopsis GUS activity 1

83 hsp70 intron MS2 coat protein 42439 rice Western 3

700282409 88 hsp70 intron MS2 coat protein 42938 rice Western 5

88 hsp70 intron MS2 coat protein 42938 wheat Elisa 4

88 hsp70 intron MS2 coat protein 42938 wheat Western 4

88 none GUS 48194 Arabidopsis GUS activity 4

88 hsp70 intron MS2 coat protein 52006 wheat Western 6

88 hsp70 intron GUS 53322 wheat GUS activity 11

700354681 90 hsp70 intron MS2 coat protein 42914 rice Western 5

90 hsp70 intron MS2 coat protein 42914 wheat Western 19

90 hsp70 intron MS2 coat protein 42936 wheat Elisa 1

90 hsp70 intron MS2 coat protein 42936 wheat Western 3

90 none GUS 51818 Arabidopsis GUS activity 7

700353007 91 hsp70 intron MS2 coat protein 52003 wheat Elisa 2

91 hsp70 intron MS2 coat protein 52003 wheat Western 2

700352625 92 hsp70 intron M52 coat protein 52021 wheat Elisa 1

[0264] 8a. 700353038

[0265] To test for anther activity in dicot plants the fragmentcomprising SEQ ID NO: 98 is places upstream of the GUS gene and put intoa plant transformation vector resulting in the construct pMON48183. Thisconstruct is used to transform Arabidopsis thaliana. Two of four showexpression in the male reproductive tissues specifically. No GUSexpression is detected in other floral tissues, stem or leaf. To testfor anther activity in monocot plants, the fragment comprising SEQ IDNO:98 is placed upstream of the hsp70 intron/MS2 coat protein genecassette and put into a plant transformation vector resulting in theconstruct pMON42438. This construct is used to transform Oryza sativum(rice). Anthers from five independent R0 rice plants are assayed byWestern Blot for MS2 coat protein. All 5 plants are positive for MS2coat protein. These data indicate that SEQ ID NO:98 can act as an antherenhanced promoter in both monocots and dicots.

[0266] 8b. 700352826

[0267] To test for anther activity in dicot plants the fragmentcomprising SEQ ID NO:94 is placed upstream of the GUS gene and put intoa plant transformation vector resulting in the construct pMON48185. Thisconstruct is used to transform Arabidopsis thaliana. Four of fourindependent events show expression in the male reproductive tissues. GUSexpression is also detected in immature seed, seedlings and cut stemsbut not detected leaves, roots or other floral organs. These dataindicate that SEQ ID NO:94 can act as an anther enhanced promoter indicots. Because it is monocot-derived promoter, it is likely to beactive in monocot anthers as well.

[0268] 8c. 700353844

[0269] To test for anther activity in dicot plants the fragmentcomprising SEQ ID NO:83 is placed upstream of the GUS gene and put intoa plant transformation vector resulting in the construct pMON48186. Thisconstruct is used to transform Arabidopsis thaliana. One event isobtained and it shows expression in the male reproductive tissuesspecifically. No GUS expression is detected in other floral tissues,leaves, or stems. To test for anther activity in monocots, the fragmentcomprising SEQ ID NO:83 is placed upstream of the hsp70 intron/MS2 coatprotein gene cassette and put into a plant transformation vectorresulting in the construct pMON42439. This construct is used totransform Oryza sativum. Anthers from three independent R0 rice plantsare assayed by Western Blot for MS2 coat protein. Two of three plantsare positive for MS2 coat protein. These data indicate that SEQ ID NO:83can act as an anther enhanced promoter in both monocots and dicots.

[0270] 8d: 700282409

[0271] To test for anther activity in dicot plants, the fragmentcomprising SEQ ID NO:88 is placed upstream of the GUS gene and put intoa plant transformation vector resulting in the construct pMON48194. Thisconstruct is used to transform Arabidopsis thaliana. Two of four eventsshow GUS expression in the anthers. One of four events has detectableGUS expression in roots but no other GUS expression is detected in othertissues.

[0272] To test for anther activity in monocot plants, the fragmentcomprising SEQ ID NO:88 is placed upstream of the hsp70 intron/MS2 coatprotein gene cassette and put into a plant transformation vectorresulting in the constructs pMON42938 and pMON52006. The constructpMON42938 is used to transform Oryza sativum (rice) and Triticumaesitivum (wheat). Anthers from five independent R0 rice plants areassayed by Western Blot for MS2 coat protein. Two of five plants arepositive for MS2 coat protein. Anthers from four independent R0 wheatplants were assayed by ELISA for MS2 coat protein. Two of four plantsare positive for MS2 coat protein. Anthers from four independent R0wheat plants are assayed by Western Blot for MS2 coat protein butexpression is below the limits of detection for each event. Theconstruct pMON52006 is used to transform wheat. Anthers from sixindependent R0 wheat plants are assayed by Western Blot for MS2 coatprotein. Three of six plants are positive for MS2 coat protein. Thesedata indicate that SEQ ID NOS: 88 can act as an anther enhanced promoterin both monocots and dicots.

[0273] 8e: 700354681

[0274] Because SEQ ID NO: 90 is a subfragment of SEQ ID NO: 87, only SEQID NO: 90 was tested. The fragment comprising SEQ ID NO:90 is placedupstream of the GUS gene and put into a plant transformation vectorresulting in the construct pMON51818. This construct is used totransform Arabidopsis thaliana. Five of seven events show GUS expressionin the anthers. GUS expression is not detected in other floral tissues.

[0275] The fragment comprising SEQ ID NO:90 is placed upstream of thehsp70 intron/MS2 coat protein gene cassette and put into a planttransformation vector resulting in the constructs pMON42914 andpMON42936. The construct pMON42914 is used to transform Oryza sativum(rice) and Triticum aesitivum (wheat). Anthers from five independent R0rice plants are assayed by Western Blot for MS2 coat protein. Four offive plants are positive for MS2 coat protein. Anthers from nineteenindependent R0 wheat plants are assayed by Western for MS2 coat protein.Fourteen of nineteen plants were positive for MS2 coat protein. Theconstruct pMON42936 is used to transform wheat. Anthers from threeindependent R0 wheat plants are assayed by Western Blot for MS2 coatprotein. One of three plants is positive for MS2 coat protein. Thesedata indicate that SEQ ID NO: 90 can act as an anther enhanced promoterin both monocots and dicots. Because SEQ ID NO: 90 acts as an antherenhanced promoter, it is probable that SEQ ID NO: 87 also acts as ananther enhanced promtoer.

[0276] 8f. 700353007

[0277] To test for anther activity in monocot plants, the fragmentcomprising SEQ ID NO:91 is placed upstream of the hsp70 intron/MS2 coatprotein gene cassette and put into a plant transformation vectorresulting in the construct pMON52003. This construct is used totransform Triticum aesitivum (wheat). Anthers from two independent R0wheat plants are assayed by Western Blot for MS2 coat protein. MS2 coatprotein is not detected in either event. Anthers from two independent R0wheat plants are assayed by ELISA for MS2 coat protein. MS2 coat proteinis not detected in either event. At this time the data obtained have notbeen able to demonstrate if this particular construct containing SEQ IDNO:91 is active in anther in transgenic wheat as the number of theevents evaluated is very low. Further evaluations are needed.

[0278] 8g: 700352625

[0279] To test for anther activity in monocot plants, the fragmentcomprising SEQ ID NO:92 is placed upstream of the hsp70 intron/MS2 coatprotein gene cassette and put into a plant transformation vectorresulting in the construct pMON52021. This construct is used totransform Triticum aesitivum (wheat). Anthers from one R0 wheat plant isassayed by Western Blot for MS2 coat protein. MS2 coat protein is notdetected. At this time the data obtained have not been able todemonstrate if this particular construct containing SEQ ID NO:92 isactive in anther in transgenic wheat as the number of the eventsevaluated is very low. Further evaluations are needed.

Example 9

[0280] Identification of Cis Elements and Engineering Novel Promoters

[0281] Cis acting regulatory elements necessary for proper promoterregulation can be identified by a number of means. In one method,deletion analysis is carried out to remove regions of the promoter andthe resulting promoter fragments are assayed for promoter activity. DNAfragments are considered necessary for promoter regulation if theactivity of the truncated promoter is altered compared to the originalpromoter fragment. Through this deletion analysis, small regions of DNAcan be identified which are necessary for positive or negativeregulation of transcription. Promoter sequence motifs can also beidentified and novel promoters engineered to contain these cis elementsfor modulating expression of operably linked transcribable sequences.See for example U.S. Pat. No. 5,223,419, herein incorporated byreference in its entirety, U.S. Pat. No. 4,990,607, herein incorporatedby reference in its entirety, and U.S. Pat. No. 5,097,025, hereinincorporated by reference in its entirety.

[0282] An alternative approach is to look for similar sequences betweenpromoters with similar expression profiles. Promoters with overlappingpatterns of activity can have common regulatory mechanisms. Severalcomputer programs can be used to identify conserved, sequence motifsbetween promoters, including, but not limited to, MEME, SIGNAL SCAN, orGENE SCAN. These motifs can represent binding sites for transcriptionfactors which act to regulate the promoters. Once the sequence motifsare identified, their function can be assayed. For example, the motifsequences can be deleted from the promoter to determine if the motif isnecessary for proper promoter function. Alternatively, the motif can beadded to a minimal promoter to test whether it is sufficient to activatetranscription. Suspected negative regulatory elements can be tested forsufficiency by adding to an active promoter and testing for a reductionin promoter activity. Some cis acting regulatory elements may requireother elements to function. Therefore, multiple elements can be testedin various combinations by any number of methods known to those skilledin the art.

[0283] Once functional promoter elements have been identified, promoterelements can be modified at the nucleotide level to affect proteinbinding. The modifications can cause either higher or lower affinitybinding which would affect the level of transcription from thatpromoter.

[0284] Promoter elements can act additively or synergistically to affectpromoter activity. In this regard, promoter elements from different 5′regulatory regions can be placed in tandem to obtain a promoter with adifferent spectrum of activity or different expression profile.Accordingly, combinations of promoter elements from heterologous sourcesor duplication of similar elements or the same element can confer ahigher level of expression of operably linked transcribable sequences.For example, a promoter element can be multimerized to increase levelsof expression specifically in the pattern affected by that promoterelement.

[0285] The technical methods needed for constructing expression vectorscontaining the novel engineered 5′ regulatory elements are known tothose of skill in the art. The engineered promoters are tested inexpression vectors and tested transiently by operably linking the novelpromoters to a suitable reporter gene such as GUS and testing in atransient plant assay. The novel promoters are operably linked to one ormore genes of interest and incorporated into a plant transformationvector along with one or more additional regulatory elements andtransformed into a target plant of interest by a suitable DNA deliverysystem. The stably transformed plants and subsequent progeny areevaluated by any number of molecular, immunodiagnostic, biochemical,phenotypic, or field methods suitable for assessing the desiredagronomic characteristic(s).

1 98 1 22 DNA Artificial Sequence Description of Artificial Sequenceadaptor sequence 1 gtaatacgac tcactatagg gc 22 2 19 DNA ArtificialSequence Description of Artificial Sequence adaptor sequence 2actatagggc acgcgtggt 19 3 30 DNA Artificial Sequence Description ofArtificial Sequence adaptor sequence 3 agggcaagct tggtcgacgg cccgggctgg30 4 17 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 4 ctgacggagg cgctacg 17 5 17 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 5 gttgaagtcgatgcagc 17 6 17 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 6 cgtcgggtat agattta 17 7 17 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 7 ccatgactca cttcctg 17 8 17 DNA Artificial Sequence Descriptionof Artificial Sequence fully synthesized primer 8 cgaatctgct acggatc 179 17 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 9 acacggtatc tctgagc 17 10 17 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 10cacacgtaat cgtaatg 17 11 17 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 11 ccatgcacca gctgcag 17 1217 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 12 cgaatctgct acggatc 17 13 17 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 13atgcgcagac gttgagg 17 14 24 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 14 ggaccccagc gtccgtagcgcctc 24 15 33 DNA Artificial Sequence Description of Artificial Sequencefully synthesized primer 15 ggatcccagc tccgacagcg agatcttacc gtc 33 1635 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 16 ggatccagat ctgtccgccg tctccgacat tagcg 35 17 35DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 17 ggatccagat ctagcgaatc tgctacggat caata 35 18 17DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 18 ggacatcacc atccagc 17 19 17 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 19catcgagcgt gccggag 17 20 27 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 20 ggatgccatc gaagctggatggtgatg 27 21 38 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 21 ggatccagat ctaagtagag agggcccaccaggtagtc 38 22 37 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 22 ggatccagat ctcccctttg ctagttctctcctcgcc 37 23 17 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 23 acgacctgga caagtac 17 24 17 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 24 tcgccttcac gttgtcg 17 25 27 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 25cagctcgccg tgtacttgtc caggtcg 27 26 39 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 26ggatccagat ctaggttgcc atccagctgg atggcgatg 39 27 27 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 27cacccggaga gcgttgtgtg cggaagc 27 28 37 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 28ggatccagat ctccttcctg tggccgccgg ctctcct 37 29 17 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 29gtggcatcat cgtcagc 17 30 20 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 30 cctcgcacag cgtcgagcag 2031 27 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 31 ctgccctcgc acagcgtcga gcagaag 27 32 39 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 32 ggatccagat cttcggcgat tgttgatgga tcggagaag 39 33 17 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 33 ccatggccaa gaagggt 17 34 21 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 34cccttctcct tgatgtccac c 21 35 26 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 35 ccttcttggc catggcgccgaacgcc 26 36 39 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 36 ggatccagat ctcgaagtgg tacgccgcgataggctcat 39 37 34 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 37 ggatccagat ctcatgtccg tagatgtgcaccac 34 38 21 DNA Artificial Sequence Description of Artificial Sequencefully synthesized primer 38 gccggcgaga gcatggcgat g 21 39 19 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 39 cgttgctgtg gtctgcttg 19 40 28 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 40gccgacacca gggtgacctc caggacac 28 41 40 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 41ggatccagat ctcatgctct cgccggcgaa ggtgttttgc 40 42 34 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 42ggatccagat ctctcgccgg cgaaggtgtt ttgc 34 43 19 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 43ctacgactag ctagattcc 19 44 18 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 44 gcggattctg ttcttgcc 1845 18 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 45 gcggattctg ttcttccc 18 46 27 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 46acgcggatcc tggtgggcgc catcgcg 27 47 39 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 47ggatccagat cttgggcgcc atcgcgcgtg gaatctagc 39 48 32 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 48ggatccagat ctcgtttgcg gtgttcgcgt tg 32 49 15 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 49ccgctttagt tcagt 15 50 15 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 50 cccgcatttc atttc 15 5127 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 51 gtctgttgtc catgcgattc acgctac 27 52 43 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 52 ggatccagat ctcatgcgat tcacgctaca gccaaatgat cga 43 53 32 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 53 ggatccagat ctgcccggtc agacatgttt ac 32 54 33 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 54ggatccagat ctgctcggcg cgttggtcgg tcg 33 55 18 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 55atgagggttc ttgtagag 18 56 18 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 56 cccatcagtc cgctgttg 1857 40 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 57 ggatcctaga tctaaacaca gagactaaca gcttctctac 40 5827 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 58 tgccgtgtga tcatattcta gagacac 27 59 27 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 59 aaacacagag actaacagct tctctac 27 60 18 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 60attctccagc gcaggtag 18 61 18 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 61 tgccctggct cgtcgaag 1862 27 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 62 cctgccacga catctttgcc cggtcag 27 63 27 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 63 tcgcacatca ggtgctcgtc cacgtac 27 64 38 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 64ggatcctaga tctctgcgct ggagaatgga tcggagag 38 65 19 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 65gaatcatcgg aataatggc 19 66 16 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 66 taggagcggg agcatc 16 6727 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 67 catcggcgga tgccatggac ctaccct 27 68 27 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 68 gccaggagga gcacgacgag gaacacg 27 69 40 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 69ggatcctaga tctgccagga ggagcacgac gaggaacacg 40 70 19 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 70caaacgctgc tgcgctctc 19 71 16 DNA Artificial Sequence Description ofArtificial Sequence fully synthesized primer 71 gagcgtggcg acgacg 16 7227 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 72 gcgccatttt ctccaggttg ttctctc 27 73 27 DNAArtificial Sequence Description of Artificial Sequence fully synthesizedprimer 73 caccgatcaa aacgacaggc tcctctg 27 74 40 DNA Artificial SequenceDescription of Artificial Sequence fully synthesized primer 74ggatcctaga tctcaccgat caaaacgaca ggctcctctg 40 75 25 DNA ArtificialSequence Description of Artificial Sequence fully synthesized primer 75caaggaggtg ttccacagcg tggcc 25 76 25 DNA Artificial Sequence Descriptionof Artificial Sequence fully synthesized primer 76 tggcgttgcg cacgacggagtgcat 25 77 27 DNA Artificial Sequence Description of ArtificialSequence fully synthesized primer 77 accgtgaagt tgtcgccggc cttcttg 27 7840 DNA Artificial Sequence Description of Artificial Sequence fullysynthesized primer 78 ggatcctaga tctacgagga tcgccgatag gccacctagg 40 792213 DNA Zea mays 79 cctgtccttc ggcatttctg tagctactct cgacgatgaggtcttcgtcg cctcatcggt 60 aggcaaagtg tcctattgca cctctaagga ggtaggactaatcaactccc tagtgacacc 120 tatgtaggcc atctgagcag aggcaatgat ctttcactaagactgattca gccaaagtta 180 actcttgaag tgctcctaaa caaataactt tggagcaaagtgaattttgg caattgttag 240 gtgaaggcag taaatgtgat ctgccctcac ctcctcagccacttttatac tgaaaatgca 300 catgacgcga cacgggtcat ggaaattgtt cccgcgcagcgtcccaagtc ccccctcgat 360 gatttaactt ctaggtcaac cgtttttact tcaaatgtatcaccttcttc ttaaacaagc 420 tttaggaagt tgtttttcgg atctttggaa ttgggcctccaagttataat ttttgcaatc 480 taagctcttt gcaaagaaaa caaactcata tcgcaatgggctacttttgg ggaccttcta 540 tgttgaagat cacttgctta aacttttact ttgacacaagctagtgtgtt tcaggaatat 600 ctctactgat gcacgaagcc aaccttcggt cgggaaggcgtgcagagaag cattgcacat 660 gaaaggtgaa agggtattaa ccaaagttga tagcttcgatgacagcaaag gagcttcaga 720 tacaagccaa aggaaaaggc gacgaaggcc taaatccgagcagccgaaga tggggaaaat 780 acgctactgc cctaacaaca tttgtaaaca gtgaggggtacaattgtaat tatgtactaa 840 gtcggttcgt ctcccctata aatagatgaa cagtaacccgcataaattac attttgccag 900 gtgctacagc tttgtatagc tcaggctcca aaacacattcgtgctatctt gcactaagaa 960 gtcaatggta tgattgtaaa cttgttttct ataagagaaatgaaattcta aggcacatga 1020 gatgagttct catatcttcg tcatgttttt atgtattctagtcgattaca tccaaccttc 1080 gtccttgagt agttatccca aagacttaac acttcaaggatgaaggcttc tactttttaa 1140 cattgtgttg tcttgttttt tatttcattt agcaattaaaagcaagtgac taacacatgg 1200 ttaaacccaa gatccgaaaa gaggctaaaa ttgagcaagaatgaacaaaa gttggtaaga 1260 ggaacataaa ccaacctttc ttagcaacat tcttccaaaaaaagaagatc aaaacatgta 1320 cccttgtatt ttgtgaaaac tggatctcca aaattgcctacaatggaagg tggctacgag 1380 aaacggttat aatcgaggag gtagagagaa ttttatgctacaaccttcac aggcggtttc 1440 cctaagaaac atccactcta aatgtctttg cacatacggttcacttaaaa aaccgcaaat 1500 gcaaattgtt cattttcact ggaggttttt taagcgaaccgctagaggaa atctcatttg 1560 caccggcgat ccttaagaca tatcatgagc gaggttgccttggaagccgg aagagttggt 1620 caatgaccta taaaaagcag aggacacagg agtgccctattcaagcattg cctaaaaata 1680 gcaaaggcca aacgaccatt tcgtgtacat agcaaacggtgctcctctct ctcaagaaag 1740 gatatcttcg ggaacatcca tccatcccca atccccaaaggcgaggagag aactagcaaa 1800 ggggaaatgg ctgcttccac aaacaacact ctcagggtgctgttcaccct aatggttgta 1860 tgcgccgcag tatgcacagc gaaaaggact gtagcaaaggcaggagactt ggcgccagcc 1920 cctgctccgt taggagcagg aggcgccacc gcagccccagaaggtgcggc tagagccagc 1980 aggacgttcg acatatcgaa gttcggcgcg accagcgacggcaagacgga ctcgacacag 2040 gttgcattgc attgcattgc attgcattgc atgttggcacggtggtgtga ctgatgcact 2100 ggttcaatga tctatcaggc agtccaggac acgtggacgtcagcgtgcgg agcgatggga 2160 gacgcaacga tgctcatccc caagggcgac tacctggtgggccctctcta ctt 2213 80 809 DNA Zea mays 80 aaaaaaccca cgggttcacgggtttgggta ctataggaac aaacccgtac caataaaccc 60 gtcgggtata gatttatgcccattaacaaa cccatggata tgaaaattga tccaaacccg 120 taccctaata gggtaaaaacccatcgggtt tcgggtttcg agtacccatt gtcatcttta 180 acaggaagtg agtcatgggcctcttgtgcg tttgcgcttc tcgcttcatg gtccgtgact 240 ttccacgggt acacatatgggccctaccat ggctctctta tcaactgggc ctcgaagcct 300 agctagttga tggcttgcataattgcattg catggtctcc tctgctccgt ccgactgagc 360 gattcttccg gtaggggagctgcagtgcag ctggtgcatg gcgatggatg gctgcgagtg 420 gtccaagaat ttctccccggcatgtcctct cctccagacc tccaccgatg cagcaggctc 480 ctggtagagc taactaaatcggggacccct tctcaagttt tcatcactat atatgcagca 540 gatacctaga agagcacgaccgagctagga gaagcgcgaa cgcgtgcatg cgcagacgtt 600 gaggtcgagg gacacggtatctctgagctt catcggagag cgacccgcca ccgccacgct 660 tggccgcaag ccgagaagagtgccgggccg ggagaccgga cgattattga tccgtagcag 720 attcgctaat ggcggatacggcggacatgg agcggatctt caagcggttc gacaccaacg 780 gcgacggtaa gatctcgctgtcggagctg 809 81 910 DNA Zea mays 81 ccgtgacttt ccacgggtac acatatgggccctaccatgg ctctcttatc aactgggcct 60 cgaagcctag ttagttgatg gcttgcataattgcattgca taattgcgct tctccctacc 120 atgtgcctgt ttgtttcggc ttctgacagcttctggccac caaaagctgc tgcggactgc 180 caaacgctct gcttttcagt cagcttctataaaattcgtt ggggcaaaaa ccatccaaaa 240 tcaatataaa cacataatcg gttgagtcgttgtaatagtt ggaatccgtc actttctaga 300 tattgaaccc tatgaacaac tttatcttcctccacacgta atcgtaatga tactcagatt 360 ctttccacag ccaaattccc ccacagccaaattttcagaa aagctggtca gaaaaaagct 420 gaaccaaaca ggcccatggt ctcctctgctccgtccggct gagcgattct tccggtggga 480 gagctgcagc tgttgcatgg cgatggatggcagcgaggtg gtccaagaat ttctccccgg 540 catgtcctct cctccagacc tccaccgatgcagcaggctc ctggtagagc taactaaatc 600 ggggacccct tctcaagttt tcatcactatatatgcagca gatacctaga agagcacgac 660 cgagctagga gaagcgcgaa cgccgtgcatgcgcagacgt tgaggtcgag ggacacggta 720 tctctgagct tcatcggaga gcgacccgccaccgccacgc ttggccgcaa gccgagaaga 780 gtgccgggcc gggagaccgg acgattattgatccgtagca gattcgctaa tggcggagac 840 ggcggacatg gagcggatct tcaagcggttcgacaccaac ggcgacggta agatctcgct 900 gtcggagctg 910 82 1511 DNA Zea mays82 atcgtgtaag gtgatttagt tcatttgttg tgagggaagt gtggtgcttg gtggtctgtt 60ccacgtggtt cctttgctcg agttttcatg ttctcatcat tcttgccttt gtttgagagg 120cgaggttatt gtcttctcca ctggagctgg gcagattttg ggttgaacca tcgcgatgta 180gctagtgaac ctgtggtctt ttgacattaa ttaaggtagt gttcggttct ggagccagtt 240gggatggagt ggctccgttc gagagatttt ggggagttgg atggctcggt atttgaatat 300aatttccctt tttagaacca ctctatcttg taaaggattg gcactcaggg ttttcttagc 360tcaaccgtca gtggaaatcg atttctgctc atggttgtct taactgaacc gccaatgaaa 420aacgattttc actagcggtt atcgtttacc tgcctatgaa aataattatt tctactggtc 480tctaacgctg gaggttctga aaaacgccag tgcaaataag tttcgaacca tctctataaa 540acttctttct actagtgaca tggaaccaat ccgaacaaca ttaaattgag agtgaagtgg 600cttgatccaa ctatagtcca gaaatcaaac actgcctaga aggctgctgg gcacgaggac 660ttgaactcta aaaaagatgt ggtgctggtg ctcttcaaaa tatttgattg ttgttcagca 720ttttgttttt gtttttgtag gcgtagttcg atttacccat ttatatgatt cgctgtgata 780caatatcata tgtaattaaa atcaactgac cccccgtttg gatcattgga attgaattcc 840attctaataa tagtaattta gatatatatc aattaagcta attcagtttt ttgcaaaata 900tatttgtata ttattattag caagatgtta gaaatattta tgtgctatat ttttactata 960gaggggtgag acgaagagtg tcttgtaagt tacagagtag aaacaaattc tactaatgca 1020taaaatcatt tctcatcctg caccccatga atttgaaccc catgaatttg agataggctt 1080atatctgaac tttgaaaagt ggtggaatgt caaatttcaa attaaataag ttaatttatt 1140aggtgaattc caattccttt gaaacaaagg gatctaaacg tcccgtgaga aaatttgcat 1200gtgcacaaaa gttcacaatt tgcatgctga cacacgcatc tctgggtccg tacgattggt 1260aaaacttgat gaggttgcct ttgtctagca tccgcatcaa taggaccttt gaaacggtaa 1320gagttggtca tcgagaacct gaaaaaaaac tagaggacag gagttcttta ttcaagcatg 1380gcctcaaaat agcaaagtcc agacggtcat ttcgtgtaaa tagcagacgg tgctcctctg 1440tctcttgcaa tcttccggaa catccatcga tctcccccca gcggcgagga gagccggcgg 1500ccacaggaag g 1511 83 459 DNA Zea mays 83 cgtatctagc gactacatgctacaacatgc tcgatgtcat atacacctat acatgtcact 60 atggcgtatc atactttgtcattaaaatcc acatctaaga catgatccat gtacaactac 120 gataagatag gagtactagttaaatctctg ttgggcattg gaccagatca tgctcgtgct 180 gggctttcgg gcctcgtgttctctcaccaa ttatagtggg tagctagtaa atgcatgcat 240 ctatatatgg acatgtatgcatgctattag agtattagtt agaagcgtac cactgcacga 300 agagagaggt acgatcgggagggaaactct catggccata cacctatcat ctccttttcg 360 tgacatccta ctgtgtatatataaccaaca acgatcatgt tagttccaca agcaaattaa 420 acctatcatc atcttctccgatccatcaac aatcgccga 459 84 1503 DNA Zea mays 84 atcctccaag gtatcagcagtgggtccgga cccccatggg aaagtgctgg acccctgttt 60 atatggaccg gacctccaggtaaggtccag gacctccacg ggcgcgaact gaaccccttg 120 gatgggtccc ggacccctctgtgtgggatc cgggccactc acaacaaggt cccgggattc 180 tgggacaaag aatacctggaccttgttgaa gaccaagcga gggtccggag ccgacacgtg 240 ttcgggccat gcggtgtacgcttctgctct ccactcaggt ggagacccga tgctgccacg 300 tggcccacca ccgtgacgtaagccagcggg cgaagcctga cgtaaggcct ctgggccaca 360 cggcctctgc atttattacagataagccgc atcgcgtgtc cactccactg gcaggcgatg 420 tgccgcctca gaatttaatgagccttgtcc actccactgg caggcgatgt gccgcctcag 480 catttaatgt gccctgtccactctgctgac aggcaacgac cagccatcct gcaggcggcg 540 tgcctgtcca ttccgttggcaagcagtacg cctatgctgc ggcatacact gtgctcatca 600 tcactcgcgt gttaccaaggaggcagcatg ggataccaat actatatgca ctacagacat 660 tatagcgctc ggggttcaccctggcgttac gggcattagt tgcttccttc catttgtccc 720 tcggcccaca tgtcggggctcagcaccctt gtacgtgccc cccttgagct ataaaaggga 780 gggcacacga cgttacaaggaagacccaac ttaggctcac acactcactc aaactcacaa 840 gttcatacaa gctctcaagctcaatacatc acacagtgga gtagggtatt acgctctggc 900 ggcccgaacc actctaaacccttgtgtgtt cttgtgttct tcccgattcc atctagcagg 960 caaaacgctt gggcccctcctcatcttagg atttagggcg ggtgcgttcc gccacccgac 1020 cggagaattc cctctccgacagtactcatg accacaaatt cagaccctgt ttgctagctc 1080 attcatcgta gcatagttccattcactcat cgaagacaaa acatggttgc gattgtgagc 1140 accatgtggt cgtcgatgcaggcgcatgtg gcgatggtcg tggcattggc gttcctagtg 1200 agcggtgact ggtgcggtcctcccaaggtt ccccctggca agaacatcac ggccacctac 1260 ggcagcgact ggctggacgctaaagcgaca tggtatggca agccaacggg tgctggcccc 1320 gacgacaacg gtggcggctgcgggtacaag gacgtgaaca agcccccctt caatagcatg 1380 ggcgcgtgca gcaacatccctatcttcaag gatggtctgg gatgtgggtc ctgcttcgag 1440 atcaagtgtg ataagcctgcggagtgctct ggcaagcccg cggtggtgta catcacggac 1500 atg 1503 85 658 DNA Zeamays 85 aaattaatac aaataaaatc atataagtca ctccttctct aaatttatcgtatatacaaa 60 attattttga ttgttattga aaattaatat acaattatat gatgcaagtttctttaatta 120 gcttattatc tatactatat aaaaatcagt atacacatgt tttatatatcatatggtact 180 tttttcatat tatagtattg atgaattttt cgcccctcta tgtcatactcctggcttcac 240 cctagtctac tacgtcaatt tttttcagta aatgcatcgc aaaatgattttgcatttttg 300 gtgtcctaaa tcttaatata tattcttaga caaatagagt taaacagatgttaaacatag 360 atttgactta agataaaaat agattttaga aaatacagaa cagccccagtattctgcatt 420 gctaaaaaac actccgtgaa acaatgtgga ccgcaaaaag ttccttcaaaatcctgccat 480 ctgatgctat ttttggggcc aaactccatc accaaccaaa cacaacctcttggctttatt 540 taacttgtgc cttgcggatg ttttcgttgt cgagggaata cgaacgtcgtacgagaacct 600 ttctccctcc tccacctttc tccttttctt gccacggcaa aacaccttcgccggcgag 658 86 1173 DNA Zea mays 86 gtcgtcaacc cggtgactgc catgggccccatgattccgc ccaccatcaa ctgcagcatg 60 accgtgctcc tacgcctgct acaaggtgcgtggagtagtc gttgctttcc tgcttgctgc 120 tcgatatgca tgccgttcgc gttgccatgcgaatgagacg aagaagaaac taaaggagga 180 tgccggcctg ttcgtggtcg caggttgcacggaggagtac agggacatct ggatgggggc 240 ggtgcatgtg cacgacgccg ccatggcgcatatcctggtg ttcgagagcc cggcggcgtc 300 cgggaggcac atctgcgccc agtccatctcccactggagc gacttcgcgg ccaaggtcgc 360 cgagctgtac cctgagtaca aggtgcccaagtaagcgacc cgaccatgtt ctgtgaaaat 420 gaaaacctgg atagatagag cattgcttagcttatagttg cgtacgttgc aggttcccca 480 aggataccca gcctgggctg gtgcgacagggagccgagga ggggtccaag aagctcgtcg 540 cgttggggct gcacttcagc cctctggagaagatcatcag ggacgctgtg gaggccctca 600 agagcagagg ctacatttcg tagctagccgaccgacggca gctatagtgg agtagtatgc 660 ctgtcgaatt tcgattccca agtggcaaattctgcaaaac gagtccgcca atatgaacaa 720 taaataaaga acgttgtgat aaaataaagcagattttctg ttgcatttgg cccttcaaag 780 catccgtggt ggtaagattt cctatgatctgtcctggtcg gtcgggcctg agcacctttt 840 ttctgtagac ggatgcttta tcacctagggattgttttat tatattgcta taatgcaaat 900 tggttgatcc aaattaaagc aggatctaaaatggtcgaca ggctaagctt ataatgaaca 960 cagaaataaa tcaaggtgga atgtgtccgcaatcgacgct gcgatttcga atgctaaata 1020 aataaatcgg taacacggac ggacgtagaagagaagccat tatgcgtggc aggcagcaca 1080 agagctattc aaagccgcgg caacggagggctgcaattca caaaccccaa aattaggtca 1140 ccccggccac tttcaacgcg aacaccgcaaacg 1173 87 1587 DNA Zea mays 87 actcttccca tttgcgagga atctccacaagttggagcct ctcaccctta caaagttatg 60 atcacaaaga aagcacaaga gtaaggatgggagagcaaca cacgcaagac tcaaatccgt 120 agcacaatca cgcacacaag ccaagacttgagctcgaaac acagcacatg gagtttgcaa 180 ctcaaacaga gctcaaatca ctaacacagcgaatcaaatg cgtggagacg gagtctggga 240 gtcttagaat gtttcttgaa agcttggtgttctgctccat gcgcctaggg gtccctttta 300 aacctccaag acagctagga gtcgttggagatcaacatgg aaggctgatc tttccttctg 360 ccgagtggcg caccggacag tccggtgcgccaccgggcag gtcctgtagc ttgtccggtg 420 tgcgatctcc ttccatatcg ggcgcatccgaccgttgagc cggcggtctc gttggcgcat 480 cagacactgt acggtgtaca ccggacagtctggtgtgccc aaccaaccgt tggcccgtcc 540 acgtgtcacc cgcagatttc gctgccgaccgttggccgtg agcgccgttg gctcatcgga 600 cagtcattag gaacgaagca taaacaaaagcgcgtgatgt ggacacacat tagggtattt 660 tgggctaact tgacacactt gagtatttataagatgtgtc atgggcttga cgactctata 720 ggctagctag cacggcacat aattaggtttgtactttagc gtgccatgct agcctatatg 780 tgtataaggc cattctcaat tgaagttttattggagtttc attctcatta aatgttgtgc 840 cacataagca aacaagacga catgtcataccatttaatga agagagatat gagagagttt 900 aatggggaat aaagctctat ttacactatttcctagacaa atattgatat ggtatccttg 960 gaattcgtat gatgaaatcc tcaattgaagaatggcttaa ctggcaccgc tacgtagggg 1020 ctattcaaga accaacaatg tacagttgttgcaacgtgaa tggttatttg cttcagatta 1080 aagccaattg tttagactga tgcagctgcaattcatagag acaaaaacag tgtagaagcc 1140 gtataagcat taagcaaaca agcgaacattgcttagctac aaccaatttg ctgggcttcc 1200 atgggcatcg cagaagtatt gtggctgcatattgctgaaa ttatagcgag ggcccaaggc 1260 ccatcacttc acttcgaggt cagcattgtacttttgttaa cgtctcgata aatttgttca 1320 cttaaaatag accagttcaa ttctggttctagtcaacatg cctggatcca cgggggagcg 1380 aggagacgaa tgtgtggccc gccgcagtgaggccaagccg agcccggtcg tccgtccaac 1440 caccccctcg tttatactat atatacacagacgcacgata cccatatcgt ggtgctagaa 1500 gcaactgaaa acagccgagc gatctcctctccctctccct ctccgatcca ttctccagcg 1560 cagcgaagta aacatgtctg accgggc 158788 665 DNA Zea mays 88 aaagaaaatt ggatgaaagt tactcgaccc cgtcaatttaacagtgacat tgaatttgtc 60 gaggctgttg agatgaagac atgccagatt gagggtattttactaataag gagattttag 120 gtgtgaatga caaacgcttc agccgaatac ttaaaagactggctaagaag aacaaagcta 180 atttggtgga ggtttcatcc aacagtgatg aggaaagaacaccaactctg gtagaagtag 240 agtatgttgc tgcatcaaag agtttttttt ttgtggaaaacttcagcgtg tagttttatt 300 ggtcagcaat gtttgtttgg cagtagcatg catgagtccgattctctgac catctccatt 360 taccggtgcc ctggttatta tccccccatc acaagagtggccaacatgca gcccctgaaa 420 cctggcgaag tccaaggggg agcgaggaga cgaacgtgtggccacggtga ggtggggatc 480 cggtccttca ccccttcaac ttgggattcc ctctctatttagccatccgt ccggtgcacg 540 atgctacaag ctcctcgtca ccagtcagaa aacagtgggatcgagttgtt tcactgcacg 600 agcacatcct ccggcgacca ccggcctccc tctccgtcctctagcgaccg accaacgcgc 660 cgagc 665 89 833 DNA Zea mays 89 acggaacctaaatatggatg tcttacaaca gctaattaga tgcgaaaggt tccagcatgc 60 ccattcgttaccctgtgaac atgggcagat ctacgggtat tatgttctgg cacaccctac 120 gtatccggtatatcttgccg attatgtttt aatactatga ggttgtttgt ataatcacat 180 ttcacaaatgagagctgaga atttaatccg tgcaaattag tttatttaat tgtttgggaa 240 tatgtttttaagtaggtgaa gataacataa ttaagatatc gattatgtct cttagtaagg 300 tctcagctaaaaagtcgtat gaactattag catgactttt cattgattta tattgtaatt 360 tatgaatatttttaacttac tttacaaatt taaggattat tatttatatt ttgaacttat 420 cctataatttaaaatttact atgtaatttc atgtaaaaat ggtttctaat ttgatcgagt 480 atatatatgaaaattttaga tgacttatag aaaaattcta gatccgccat tggctgcaga 540 gtgtagaggatgtgcatgca cagatgcact tcattgttgt tatatataca acaagttttc 600 atgcaatacaagcctataaa taaatgtcct gactaagctt tcgtccacag aatttaccac 660 ttcttccgctgagtactacc gattcaacag aacagataga ccactcgtta acactgtaca 720 cttctacctatatattcgct tctctcctct tgcaaatcat attgtcaata gtaacagtga 780 gaagaacacacaaaatgagg gttcttgtag agaagctgtt agtctctgtg ttt 833 90 823 DNA Zea mays90 ctgcacggta ctccaagtat aagacacagc taaaacacaa cataatgcag tggtcatgtc 60taaaacatgt gtcttaccat attcattgta tcaatcagaa cattcaataa attaaagtga 120ccaatcagat agtctcctgt cccgaatata gagctaagac actgtgtctt cgtcaagata 180catgtcttga gattttttac attcaccccc ctagacacac tctaagacac aacttaagac 240acccattgta catgccctaa ctggcaccgc tacgtagggg ctattcaaga accaaccatg 300tacagttgtt gcaacgtgaa tggttatttg cttcagatta aagctaatta tttagactga 360tgcagctgca attcatagag acaaaaacag tgtagaagcc gtataagcat taagcaaaca 420agcgaacatt gcttagctac aaccaatttg ctgggcttcc atgggcatcg cagaagtatt 480gtggctgcat attgctgaaa ttatagcgag ggcccaaggc ccatcacttc acttcgaggt 540cagcattgta cttttgttaa cgtctcgata aatttgttca cttaaaatag accagttcaa 600ttctggttct agtcaacatg cctggatcca cgggggagcg aggagacgaa tgtgtggccc 660gccgcagtga ggccaagccg agcccggtcg tccgtccaac caccccctcg tttatactat 720atatacacag acgcacgata cccatatcgt ggtgctagaa gcaactgaaa acagccgagc 780gatctcctct ccctctccct ctccgatcca ttctccagcg cag 823 91 1163 DNA Zea mays91 actacagccc gagggcgcct gtctacgggc ccctcggcgc agactatctg gttgtcccac 60cggatagtcc ggtgcacacc agacaattac tgttcactgt ccggtgtgcc atcaggcgtt 120ggttgactgc ctttttcttg gatttcttcg cagtttcttt tgagcttctt ttgttcttga 180gtcttggact tctatgcttc tttttatatc ttcttttgag gtgttgcatc ctcattgcct 240tagtccaatc ctcttcgcat cctgtgaact acaaacataa acactagcaa acacattagt 300ccacaggttg cgttgttcat caaacaccaa aaccttttga gccaaatggc acaggtccat 360tttcattaca gccaccctcc tcagtcgttg gttgttagtt attttcgacg gctcacgtgt 420atagccgcca aaatttggca aatttcggca ccacaatgtc caaccatcga aaattagaca 480atagggaaaa tcacgggccg ccctttcatt ttccacggcg aattagggtc accaaaccaa 540aacaatcgaa aaccaaacca cgagccttaa tttttgtggc cttgaccgcc aaaaattaca 600tgttttcttc tagtggtagg gggagttata agcaacaact ctaacaattg tagaaaaata 660acattgggtg accaagatga gtaagagagg aatttaggat gagattaata tgtgtttatt 720gctatctaaa ctttatacat gaggtttcta ggctcgtcat atgttataga gtcaaaaagt 780atgacatgtt tttttagtca caacaaagtg tggctttcca cacttttgtg gtttatcttg 840tttaactaag attagccatg acaatttatg agcactcgca tgtttggcca cctatatata 900gcgagacttg tgcatccaag acttcttccg tgcgagggta gtgcacgacc ataggacaag 960aggagcttgc attcgcgcgt ctcaaggcaa caatctcccc taaaaatagc cacacaacat 1020tcatgttgcc tatatataaa catcgtgcct cgcccgtccc atcatcacag tcgaaacaaa 1080gccacaacac atacaggaaa gcaagcaaga atcatcggaa taatggctcg tgcatgcgtg 1140ttcctcgtcg tgctcctcct ggc 1163 92 2126 DNA Zea mays 92 cctccgaaatcaccgaccac agagatacac ttgcacgggt gtgcgggcga tcagattttt 60 ggggagcgtcttcgcgactg ctcgcgtgat cgtccacagc ttgctgttcg tcgccttccc 120 aagttgacgcgtgctgctgt tcttcttccc ggcgaccgtt cgagggactg cactgcgtac 180 atcttcctgcaccgacttcg tacggctaca tcgaacaaac acacgagatg tctcgtgtga 240 atggagccactggtgccttg agcatcggtc cctccgctgg gtacactctg ttcttcgtat 300 ttgtgcatgtttcattgctg tttactgctt atgcgagtag ttatacacat atgcacatac 360 atgtcatcacatatatcgca ctgattatct ggattaaatt aaaactaaaa atgcctaact 420 ttctaacaatatttgcactt gttcttacta ttcctgtttg atttggtttt ttgattgagt 480 gtgatgagttgtgaagtaat gtctataatt gcatctaaat atatatatat gcataaaaat 540 atagaaatactcccataaaa acagatccat cttatcttga aagattctat attatcctaa 600 tagatccatcttgtcctgat aagcatactt attccacctt agagtaacaa tcatgatcta 660 atccaaattaattagatcta atctaatcta atctaattca atataatcta atttgaccta 720 atttagtcaaaactagtcta atctaatctg aaactcttat cttattgatc tttcttgcct 780 atatctaaaggttagaacta attaacttat ctagtccaac ctaggagcaa aacaacaaac 840 atgattctacatattctcat gaagcttaag ccacctatta agccatatgc tctacctact 900 gagctatttggtgtacctac aaacccattt taaccataaa tcatttaatt tgcaattagt 960 gaacattgtaattgtcctag ttgcctggtt tgttgtatta tatatttgag taacgagtaa 1020 tgacttcatatttggattga gctagtaact taactaattc caacttggat atggatgtat 1080 atatggttgtatccgtgggg gtggcctttg atttggttat atttcacttg agagagtagt 1140 attgtaatgttctgaagagt tgttcagtat tttctggaac aaagaagata aatacttccg 1200 taaggttgcacaacttatca tttgaaaggg gttactatgt ttggttgtga tgcatatagc 1260 tcatgctgaaactaatcatc tttgttgatt agaacggcta ggaggttagc agttgtagaa 1320 gttgcagtaattacagaaaa aaaaaggatg gatggaagca atccaatatg tgcatgcaaa 1380 atttgggtttgaacaaggac ttatggtaag agaaacatgc gcttgaacag tatgtgtagg 1440 agactcaaaggagttctgga gagagaggtt tcttcttttt tttcttagtt ttgacttccg 1500 gtctataactaacattgtaa gagcaaaaat gagaaaaaaa catgactaaa gttaaaaata 1560 aggacaccagcaaaaccaaa ggcaccacct taaaaaactg ttttcttttt ctgaacacag 1620 aatcaaggggatttatttgt tattaatact aacagaaaat gttagcagga gtgacagtct 1680 gttggagcaggattctgcga ggaagccgct tatccataga caggaatttt tttagtgata 1740 agtagggcacactcttaact ttgcatgagg agtttgggta ccaaatacag gtgaggggtg 1800 aggcttcgtcagtcgttgca caggtaaaga ttgatgattt gatgtaactt tgaaccgctc 1860 taactaactaaatcgtcctc gagtgtgcgg cgggtgcgag caacaaggcc gtccgctcct 1920 tgttcgtccctgcatgtgtt ccgttcggtt ccatcaattc caccacgaaa taaggctgta 1980 taaatctttcctgggcgttc cctctctctc ctgtgcatcg ccggacggaa ccaaacgcca 2040 aacgctgctgcgctctctcc ttctcgtcct gaccccccag agcgagaggg aggggcaccc 2100 agaggagcctgtcgttttga tcggtg 2126 93 2508 DNA Zea mays 93 actgcttgtg aaaagtaaactgaattctgg ttgtgaacta cttgtttaag taaatgcgtg 60 tttctgtttt ttgttgtcagtgcatttctg ttttcactga tgaaaccacc atttctgctt 120 tcaatgaatc tattgaactgaactgcaaaa aaagaaattg ttctattttg tttgagtgca 180 caaacggaac tcaacggaactagatctgaa tatgtttgag tgcacaaaca gaaattgttc 240 tgttctggtt tcagtgaactccacaaatag aactgaatct attatgtttg agtgtagaaa 300 cagataccga aatgctacatctagcactta atctggcaga atcagaaaat tggggcaaat 360 acaagttgtt taagagcacaaacagaaact gaatctgttt gattgcagaa accaataaaa 420 acagaaatat atgttgaaaataattcataa gtaggcagtg gtggtgctat ggtgcatacc 480 aggttcttat tcaaatgatttgctaaagtc aaatatatgc ttctggtctg attgatttgt 540 gaaactgaaa tggatattttatttcggcac tatgaaataa actcactgtg atcctgaaac 600 atatcagttg tgtttgtttttgtaaatctt ttataccact aggggagaaa attagcttag 660 ttcaatcgca tctcatatgtctaattacca ggggagaaaa ttagcttagt tcattttgtt 720 gctgccatat gggtgaaaaaataatgagac atctaaatca gtaaattgga aatatagcat 780 cttaaacctg caggtagtttcttaaacctg attctagcta caacttagta caactaccgg 840 tagtttttta aacctgattctagctacatg ttttatattg tggcacaaga acttttaaga 900 acatatgctg atgcccactgtatttagtta ctacttcaag accaactgta ttttagttac 960 aaatgtgttt tcaagattatagaaatttgt agctgaaatt atccacacca tatttgtgaa 1020 ctgacatcat ttctaagaatattactgatt agaatctttc acttttataa tgctttgcag 1080 gagtggcccc tctggagttgaatatgcagt tataaccaaa ttttacccct tttatcctag 1140 aagagttgcc aagacacggtataagaccat gataatagac taagagagga tttggctcta 1200 attactatat gttttatttatgcagtccca tgagaacttt gagtatttgc agattgcttt 1260 attaatttat taaagttaaagattgtatgt gttgagtatg tatccactct tgttggaagt 1320 gtcttgcaat tccaatccaaggatgtataa aatactgcat gggctaagta tgtgtttttt 1380 catgtatttg gagtatatatactttttgtt gcttgagaac atgtatgtac actagaagct 1440 tgtcaattgt gtgaacttgagttgatccct gtctaacctg agtatatata tatatatata 1500 ttttgttgct tgagaacaagtatgtacaat agaggcttga caattgtgtg aacttgagtt 1560 gaacatgaat tttgataatcacaactcacc atccctttca atatgcttag aatatagctt 1620 tttataattt ttcaccctacaatacaaaat tgttctatga aggccatggt acatcatcat 1680 atcctgtatt atcaacctaggatttgtcta tttcgattaa taatggcatt gagtcaaatt 1740 ttggttgttt caaatgatagacttcgatat ttgttatgat ttatgagttg attcttgata 1800 gcattactaa aaaatgacctatgtatatac aagtgtcttc cgttgcaacg cacggacata 1860 tacctagtca atcactaagaccctaatttt gaagttggga cttagacgtg ttccacgttt 1920 gtaaaggcaa gtatataggtgtatgtatat aagagccggt gtatacaaca attttttata 1980 agaaaacttg aacaagtagccaggtgttga aatcttcata tatgtgccga cgccattcaa 2040 catcatattt ggcttctggcgaggatcgta gtatcaagca acataaaagc aatgacaaac 2100 agcgaagcac aaagatctcccaggctcgtc ataaactaat cacaatgttg tttgtcctcc 2160 acaattagca caacccattttagaaaaaga tgccacgatc gatcgagacg ttggccagct 2220 atcaaacaga taagaactacccaaatattt cctaaatcca gaacggaaga cccattgact 2280 aggtccttac ctctcaaatagacagactat tcttctccac atcaaaatat agggactccc 2340 gatgcaacaa acacgggccaccacacaaca atggtgaaat gaccatgcat gcatccacgt 2400 ccgtacgcag ccatttcgtctataaatttg cttcccatcc gattcaacta caagcttgcg 2460 ggcaaaaatg gcaaaggctctcctaggtgg cctatcggcg atcctcgt 2508 94 1797 DNA Zea mays 94 aagctttaggaagttgtttt tcggatcttt ggaattgggc ctccaagtta taatttttgc 60 aatctaagctctttgcaaag aaaacaaact catatcgcaa tgggctactt ttggggacct 120 tctatgttgaagatcacttg cttaaacttt tactttgaca caagctagtg tgtttcagga 180 atatctctactgatgcacga agccaacctt cggtcgggaa ggcgtgcaga gaagcattgc 240 acatgaaaggtgaaagggta ttaaccaaag ttgatagctt cgatgacagc aaaggagctt 300 cagatacaagccaaaggaaa aggcgacgaa ggcctaaatc cgagcagccg aagatgggga 360 aaatacgctactgccctaac aacatttgta aacagtgagg ggtacaattg taattatgta 420 ctaagtcggttcgtctcccc tataaataga tgaacagtaa cccgcataaa ttacattttg 480 ccaggtgctacagctttgta tagctcaggc tccaaaacac attcgtgcta tcttgcacta 540 agaagtcaatggtatgattg taaacttgtt ttctataaga gaaatgaaat tctaaggcac 600 atgagatgagttctcatatc ttcgtcatgt ttttatgtat tctagtcgat tacatccaac 660 cttcgtccttgagtagttat cccaaagact taacacttca aggatgaagg cttctacttt 720 ttaacattgtgttgtcttgt tttttatttc atttagcaat taaaagcaag tgactaacac 780 atggttaaacccaagatccg aaaagaggct aaaattgagc aagaatgaac aaaagttggt 840 aagaggaacataaaccaacc tttcttagca acattcttcc aaaaaaagaa gatcaaaaca 900 tgtacccttgtattttgtga aaactggatc tccaaaattg cctacaatgg aaggtggcta 960 cgagaaacggttataatcga ggaggtagag agaattttat gctacaacct tcacaggcgg 1020 tttccctaagaaacatccac tctaaatgtc tttgcacata cggttcactt aaaaaaccgc 1080 aaatgcaaattgttcatttt cactggaggt tttttaagcg aaccgctaga ggaaatctca 1140 tttgcaccggcgatccttaa gacatatcat gagcgaggtt gccttggaag ccggaagagt 1200 tggtcaatgacctataaaaa gcagaggaca caggagtgcc ctattcaagc attgcctaaa 1260 aatagcaaaggccaaacgac catttcgtgt acatagcaaa cggtgctcct ctctctcaag 1320 aaaggatatcttcgggaaca tccatccatc cccaatcccc aaaggcgagg agagaactag 1380 caaaggggaaatggctgctt ccacaaacaa cactctcagg gtgctgttca ccctaatggt 1440 tgtatgcgccgcagtatgca cagcgaaaag gactgtagca aaggcaggag acttggcgcc 1500 agcccctgctccgttaggag caggaggcgc caccgcagcc ccagaaggtg cggctagagc 1560 cagcaggacgttcgacatat cgaagttcgg cgcgaccagc gacggcaaga cggactcgac 1620 acaggttgcattgcattgca ttgcattgca ttgcatgttg gcacggtggt gtgactgatg 1680 cactggttcaatgatctatc aggcagtcca ggacacgtgg acgtcagcgt gcggagcgat 1740 gggagacgcaacgatgctca tccccaaggg cgactacctg gtgggccctc tctactt 1797 95 828 DNA Zeamays 95 ccgtgacttt ccacgggtac acatatgggc cctaccatgg ctctcttatcaactgggcct 60 cgaagcctag ttagttgatg gcttgcataa ttgcattgca taattgcgcttctccctacc 120 atgtgcctgt ttgtttcggc ttctgacagc ttctggccac caaaagctgctgcggactgc 180 caaacgctct gcttttcagt cagcttctat aaaattcgtt ggggcaaaaaccatccaaaa 240 tcaatataaa cacataatcg gttgagtcgt tgtaatagtt ggaatccgtcactttctaga 300 tattgaaccc tatgaacaac tttatcttcc tccacacgta atcgtaatgatactcagatt 360 ctttccacag ccaaattccc ccacagccaa attttcagaa aagctggtcagaaaaaagct 420 gaaccaaaca ggcccatggt ctcctctgct ccgtccggct gagcgattcttccggtggga 480 gagctgcagc tgttgcatgg cgatggatgg cagcgaggtg gtccaagaatttctccccgg 540 catgtcctct cctccagacc tccaccgatg cagcaggctc ctggtagagctaactaaatc 600 ggggacccct tctcaagttt tcatcactat atatgcagca gatacctagaagagcacgac 660 cgagctagga gaagcgcgaa cgccgtgcat gcgcagacgt tgaggtcgagggacacggta 720 tctctgagct tcatcggaga gcgacccgcc accgccacgc ttggccgcaagccgagaaga 780 gtgccgggcc gggagaccgg acgattattg atccgtagca gattcgct 82896 847 DNA Zea mays 96 ccgtgacttt ccacgggtac acatatgggc cctaccatggctctcttatc aactgggcct 60 cgaagcctag ttagttgatg gcttgcataa ttgcattgcataattgcgct tctccctacc 120 atgtgcctgt ttgtttcggc ttctgacagc ttctggccaccaaaagctgc tgcggactgc 180 caaacgctct gcttttcagt cagcttctat aaaattcgttggggcaaaaa ccatccaaaa 240 tcaatataaa cacataatcg gttgagtcgt tgtaatagttggaatccgtc actttctaga 300 tattgaaccc tatgaacaac tttatcttcc tccacacgtaatcgtaatga tactcagatt 360 ctttccacag ccaaattccc ccacagccaa attttcagaaaagctggtca gaaaaaagct 420 gaaccaaaca ggcccatggt ctcctctgct ccgtccggctgagcgattct tccggtggga 480 gagctgcagc tgttgcatgg cgatggatgg cagcgaggtggtccaagaat ttctccccgg 540 catgtcctct cctccagacc tccaccgatg cagcaggctcctggtagagc taactaaatc 600 ggggacccct tctcaagttt tcatcactat atatgcagcagatacctaga agagcacgac 660 cgagctagga gaagcgcgaa cgccgtgcat gcgcagacgttgaggtcgag ggacacggta 720 tctctgagct tcatcggaga gcgacccgcc accgccacgcttggccgcaa gccgagaaga 780 gtgccgggcc gggagaccgg acgattattg atccgtagcagattcgctaa tggcggagac 840 ggcggac 847 97 727 DNA Zea mays 97 aaaaaacccacgggttcacg ggtttgggta ctataggaac aaacccgtac caataaaccc 60 gtcgggtatagatttatgcc cattaacaaa cccatggata tgaaaattga tccaaacccg 120 taccctaatagggtaaaaac ccatcgggtt tcgggtttcg agtacccatt gtcatcttta 180 acaggaagtgagtcatgggc ctcttgtgcg tttgcgcttc tcgcttcatg gtccgtgact 240 ttccacgggtacacatatgg gccctaccat ggctctctta tcaactgggc ctcgaagcct 300 agctagttgatggcttgcat aattgcattg catggtctcc tctgctccgt ccgactgagc 360 gattcttccggtaggggagc tgcagtgcag ctggtgcatg gcgatggatg gctgcgagtg 420 gtccaagaatttctccccgg catgtcctct cctccagacc tccaccgatg cagcaggctc 480 ctggtagagctaactaaatc ggggacccct tctcaagttt tcatcactat atatgcagca 540 gatacctagaagagcacgac cgagctagga gaagcgcgaa cgcgtgcatg cgcagacgtt 600 gaggtcgagggacacggtat ctctgagctt catcggagag cgacccgcca ccgccacgct 660 tggccgcaagccgagaagag tgccgggccg ggagaccgga cgattattga tccgtagcag 720 attcgct 72798 746 DNA Zea mays 98 aaaaaaccca cgggttcacg ggtttgggta ctataggaacaaacccgtac caataaaccc 60 gtcgggtata gatttatgcc cattaacaaa cccatggatatgaaaattga tccaaacccg 120 taccctaata gggtaaaaac ccatcgggtt tcgggtttcgagtacccatt gtcatcttta 180 acaggaagtg agtcatgggc ctcttgtgcg tttgcgcttctcgcttcatg gtccgtgact 240 ttccacgggt acacatatgg gccctaccat ggctctcttatcaactgggc ctcgaagcct 300 agctagttga tggcttgcat aattgcattg catggtctcctctgctccgt ccgactgagc 360 gattcttccg gtaggggagc tgcagtgcag ctggtgcatggcgatggatg gctgcgagtg 420 gtccaagaat ttctccccgg catgtcctct cctccagacctccaccgatg cagcaggctc 480 ctggtagagc taactaaatc ggggacccct tctcaagttttcatcactat atatgcagca 540 gatacctaga agagcacgac cgagctagga gaagcgcgaacgcgtgcatg cgcagacgtt 600 gaggtcgagg gacacggtat ctctgagctt catcggagagcgacccgcca ccgccacgct 660 tggccgcaag ccgagaagag tgccgggccg ggagaccggacgattattga tccgtagcag 720 attcgctaat ggcggatacg gcggac 746

What is claimed is:
 1. An isolated nucleic acid comprising a sequenceselected from the group consisting of SEQ ID NOS:79-98, or a fragment,region, or cis element of said sequence thereof, said isolated nucleicacid being capable of regulating transcription of an operably linked DNAsequence.
 2. The isolated nucleic acid of claim 1 wherein the isolatednucleic acid is a promoter.
 3. The isolated nucleic acid of claim 2wherein the promoter is a hybrid promoter.
 4. The isolated nucleic acidof claim 3 wherein said isolated nucleic acid confers enhancedexpression of operably linked genes in male reproductive tissues.
 5. Theisolated nucleic acid of claim 4 wherein said isolated nucleic acidconfers enhanced expression of operably linked genes in anthers.
 6. Theisolated nucleic acid of claim 5 wherein said isolated nucleic acidconfers enhanced expression of operably linked genes in wheat anthers.7. The isolated nucleic acid of claim 4 further comprising a minimalpromoter.
 8. The isolated nucleic acid of claim 7 wherein the minimalpromoter is selected from the group consisting of a minimal CaMV and arice actin promoter.
 9. The isolated nucleic acid of claim 8 wherein theminimal promoter is a minimal CaMV 35S promoter.
 10. A promotercomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOS: 79-98 and fragments thereof.
 11. The promoter of claim 10wherein said promoter confers enhanced expression of operably linkedgenes in male reproductive tissues.
 12. The promoter of claim 11 whereinsaid promoter confers enhanced expression of operably linked genes inanthers.
 13. The promoter of claim 12 wherein said promoter confersenhanced expression of operably linked genes in wheat anthers.
 14. Acell comprising a DNA construct comprising an isolated nucleic acidsequence selected from the group consisting of SEQ ID NOS:79-98 or afragment, region, or cis element of said sequence thereof, and operablylinked to said nucleic acid sequence, a transcribable DNA sequence and a3′ non-translated region.
 15. A transgenic plant comprising a DNAconstruct comprising an isolated nucleic acid sequence selected from thegroup consisting of SEQ ID NOS:79-98 or a fragment, region, or ciselement of said sequence thereof, and operably linked to said nucleicacid sequence, a transcribable DNA sequence and a 3′ non-translatedregion.
 16. A method of regulating transcription of a DNA sequencecomprising operably linking the DNA sequence to a promoter comprising anucleic acid sequence selected from the group consisting of SEQ IDNOS:79-98.
 17. The method of claim 16 comprising operably linking theDNA sequence to a hybrid promoter comprising the nucleic acid sequenceselected from the group consisting of SEQ ID NOS:79-98.
 18. The methodof claim 16 wherein operably linking the nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS:79-98 or fragment thereof to thepromoter confers enhanced expression of operably linked genes in malereproductive tissues.
 19. The method of claim 18 wherein said malereproductive tissues comprise monocot or dicot male reproductivetissues.
 20. The method of claim 19 wherein said male reproductivetissues comprise anthers.
 21. The method of claim 20 wherein said malereproductive tissues comprise wheat anthers.
 22. The method of claim 16comprising operably linking a minimal promoter to the nucleic acidsequences selected from the group consisting of SEQ ID NOS:79-98 orfragment, region, or cis element thereof.
 23. A method of making atransgenic plant comprising introducing into a cell of a plant a DNAconstruct comprising: (i) a promoter comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOS:79-98 or a fragment,region or cis element thereof, and, operably linked to the promoter,(ii) a transcribable DNA sequence and (iii) a 3′ non-translated region.24. A method of isolating at least two 5′ regulatory sequences thatconfer enhanced expression of operably linked genes in male reproductivetissues from a plant comprising: (i) evaluating a collection of nucleicacid sequences of ESTs derived from at least one cDNA library preparedfrom a plant cell type of interest; (ii) comparing EST sequences from atleast one target plant cDNA library and at least one non-target cDNAlibraries of ESTs from a different plant cell type; (iii) subtractingcommon EST sequences found in both target and non-target libraries; (iv)designing gene specific primers from the remaining EST sequences aftersaid subtraction; and (v) isolating the corresponding 5′ flanking andregulatory sequences from a genomic library prepared from the targetplant comprising the use of said primers.
 25. The method of claim 24wherein said male reproductive tissues are from monocot or dicot plants.26. The method of claim 25 wherein said male reproductive tissuescomprise anthers.
 27. The method of claim 26 wherein said malereproductive tissues comprise wheat anthers.